Polynucleotides encoding BREX system polypeptides and methods of using same

ABSTRACT

Isolated polynucleotides encoding a BREX system are provided. Accordingly there is provided an isolated polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that said BREX system does not comprise pglW, and wherein said BREX system confers phage resistance to a bacteria recombinantly expressing same; Also provided is an isolated polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, and wherein said BREX system confers phage resistance to a bacteria recombinantly expressing same. Also provided are compositions and methods for conferring phage resistance to bacteria or for conferring bacterial susceptibility to phages.

RELATED APPLICATIONS

This application is a National Phase of PCT Patent Application No.PCT/IL2014/050902 having International filing date of Oct. 14, 2014,which claims the benefit of priority under 35 USC § 119(e) of U.S.Provisional Patent Application No. 61/894,993 filed on Oct. 24, 2013.The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 65640SequenceListing.txt, created on Apr. 21,2016, comprising 61,303,214 bytes, submitted concurrently with thefiling of this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates topolynucleotides encoding BREX system polypeptides and methods of usingsame.

The ongoing arms race between bacteria and bacteriophages (phages) hasled to the rapid evolution of efficient resistance systems to protectbacteria from phage infection (Stern and Sorek, 2011). These systemsinclude restriction-modification systems enzymes that recognize andcleave foreign DNA (King and Murray, 1994), abortive infection (Abi)mechanisms that lead to the suicide of the infected host, thusprotecting the colony against phage spread (Chopin et al., 2005), andthe CRISPR/Cas adaptive defense system, which uses small RNAs to targetand destroy invading phage DNA (Deveau et al., 2010). On the counterarm, as part of this continuous bacteria and phages arms race,successful phages had also developed numerous counter-resistancemechanisms to overcome bacterial defense (Stern and Sorek, 2011). Due tothe rapid evolution and elaborated biological novelty associated withthe bacteria-phage arms race, it is estimated that many additional, yetuncharacterized anti-phage defense systems are encoded by bacteria andarchaea genomes (Stern and Sorek, 2011).

A broad array of food products, commodity chemicals, and biotechnologyproducts are manufactured industrially by large-scale bacterialfermentation of various substrates. Enormous amounts of bacteria arebeing cultivated each day in large fermentation vats, thus phagecontamination can rapidly bring fermentations to a halt and causeeconomic setbacks, and is therefore considered a serious threat in theseindustries. The dairy fermentation industry has openly acknowledged theproblem of phage and has been working with academia and starter culturecompanies to develop defense strategies and systems to curtail thepropagation and evolution of phages for decades.

Anti-microbial phage therapy dates back to the early 1900s, after theirco-discovery by Frederick Twort and Felix d'Hérelle (Twort F W 1915; andD'Hérelle 1917). Over the last decade a marked increase in interest inthe therapeutic use of phages has been observed, which has resulted dueto a substantial rise in the prevalence of antibiotic resistance ofbacteria, coupled with an inadequate number of new antibiotics(Miedzybrodzki R et al., 2012). Properly formulated and applied phageshave sufficient potential to cure bacterial infections. The keyadvantage of phages as anti-microbial therapeutic agents is theirpotential to negatively impact only their specific bacterial targets.Other advantages include, for example, an increase in phage number overthe course of treatment, tendency to only minimally disrupt normalflora, capability of disrupting bacterial biofilms, low inherenttoxicities, and most importantly effectiveness against bothantibiotic-sensitive and antibiotic-resistant bacteria.

In 1982, Chinenova and colleagues reported a unique phage defensephenotype in Streptomyces coelicolor A3(2), which was denoted PhageGrowth Limitation (PGL) (Chinenova T. A. et al, 1982). In their workChinenova et al. demonstrated that upon the first cycle of infection bythe ΦC31 phage, Streptomyces coelicolor A3 was phage-sensitive andsupported phage burst. However, phages emerging from this first cycle ofinfection could not successfully re-infect the Streptomyces coelicolorA3 host. Intriguingly, these phages were able to successfully infectstrains of Streptomyces that do not carry the PGL system (Chinenova T.A. et al, 1982).

Further studies mapped the phenotype to a cluster of four genes, denotedpglW, pglX, pglY and pglZ, which were shown to reconstitute the abovedescribed PGL phenotype upon transfer to a PGL host (Sumby. P. & Smith,M. C. 2002). Of note, introduction of pglY and pglZ⁻ was not sufficientto confer a PGL+ phenotype in all mutants tested (Laity et al., 1993;Sumby et al. 2002). The domains encoded within these four genes do notresemble any classical combination of genes currently known to beinvolved in phage defense: pglZ is a member of the alkaline phosphatasesuperfamily; pglW has a serine/threonine kinase domain; pglX is anadenine-specific DNA methyltransferase; and pglY contains a p-loopATPase domain (Sumby, P. & Smith, M. C. 2002). The PGL system describedto date was not active against any other phage except for ΦC31 and itshomoimmune relatives (Sumby. P. & Smith, M. C. 2002; Laity, C. et al.1993).

A major characteristic of the PGL system described to date is theinitial release of phage from the first infectious cycle followed by theattenuation of phage growth in the second. Various combinations of genesbelonging to the PGL system, and predominantly pglZ, were found to beenriched within ‘defense islands’ (typical clustering of genes encodingdefense system components in microbial genomes), providing additionalsupport to the general involvement of these genes in a complexanti-phage defense system in multiple species (Makarova, K. S. et al.2011; Makarova, K. S. et al. 2013). The discovery of the PGL system asan additional line of defense in bacteria may shed more light on thecomplex bacteria and phage arms race. However, a molecular mechanismthat explains the activity of the PGL system has not yet been solved.Profound understanding of the molecular mechanism of this system mightprove to be a powerful and economically important tool in molecularengineering applications (as was previously demonstrated with othercomplex phage resistance systems such as CRISPR-Cas).

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide encoding a BREX systemcomprising a nucleic acid sequence encoding the BREX system comprisingbrxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII,brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREXsystem does not comprise pglW, and wherein the BREX system confers phageresistance to a bacteria recombinantly expressing same.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide encoding a BREX systemcomprising a nucleic acid sequence encoding the BREX system comprisingbrxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, andwherein the BREX system confers phage resistance to a bacteriarecombinantly expressing same.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide comprising a nucleic acidsequence encoding a BREX system comprising:

-   -   (i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL;    -   (ii) brxA, brxB, (brxC/pglY)_(x2), pglX, pglZ and brxHII;    -   (iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI;    -   (iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA;    -   (v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHI; or    -   (vi) brxP, brxC/pglY, pglZ and brxL.

According to some embodiments of the invention, the nucleic acidconstruct comprising the polynucleotide encoding the BREX system furthercomprises a cis-acting regulatory element for directing expression ofthe nucleic acid sequence.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct system comprising at leasttwo nucleic acid constructs expressing a BREX system comprisingbrxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII,brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREXsystem does not comprise pglW.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct system comprising at leasttwo nucleic acid constructs expressing a BREX system comprisingbrxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI.

According to an aspect of some embodiments of the present inventionthere is provided a phage defense composition, comprising as an activeingredient a BREX system comprising brxC/pglY, pglZ and at least one ofpglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, andbrxE, with the proviso that the BREX system does not comprise pglW, orcomprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD andbrxHI; and an acceptable carrier or diluent.

According to some embodiments of the invention, the nucleic acidconstruct or the composition comprises the BREX system formulated in aformulation suitable for cell penetration.

According to an aspect of some embodiments of the present inventionthere is provided an isolated cell genetically modified to express aBREX system selected from the group consisting of

(1) brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI,brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso thatthe BREX system does not comprise pglW,

(2) brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI.

(3i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL,

(3ii) brxA, brxB, (brxC/pglY)_(x2), pglX, pglZ and brxHII,

(3iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI,

(3iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA,

(3v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHI, or

(3vi) brxP, brxC/pglY, pglZ and brxL.

According to some embodiments of the invention, the genetically modifiedcell further being resistant to a first cycle phage infection.

According to some embodiments of the invention, the genetically modifiedcell being resistant to phage lysogeny.

According to some embodiments of the invention, the genetically modifiedcell being resistant to lytic phage.

According to some embodiments of the invention, the genetically modifiedcell being resistant to phage DNA replication.

According to an aspect of some embodiments of the present inventionthere is provided an isolated cell genetically modified to express aBREX system polypeptide selected from the group consisting of pglXI,brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE.

According to some embodiments of the invention, the isolated cell doesnot express a BREX system endogenously.

According to some embodiments of the invention, there is provided amethod of protecting bacteria from phage attack, the method comprisingexpressing in the bacteria the isolated polynucleotide or the nucleicacid construct, thereby protecting the bacteria from phage attack.

According to an aspect of some embodiments of the present inventionthere is provided a method of protecting first bacteria from phageattack, the method comprising contacting the first bacteria with secondbacteria which expresses on a transmissible genetic element a BREXsystem comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP,brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the provisothat the BREX system does not comprise pglW, or comprising brxC/pglY,pglZ, pglX, pglW and at least one of brxD and brxHI, wherein the firstbacteria and the second bacteria are non identical; thereby protectingthe bacteria from phage attack.

According to some embodiments of the invention, the first bacteria doesnot express a BREX system endogenously.

According to some embodiments of the invention, the bacteria does notexpress a BREX system endogenously.

According to some embodiments of the invention, the phage is selectedfrom the group consisting of SPβ, SP16, Zeta, Φ3T and SPO2.

According to some embodiments of the invention, the phage is not Φ105,rho10 and rho14.

According to some embodiments of the invention, the phage is a lyticphage.

According to some embodiments of the invention, the lytic phage is SPO1and/or SP82G.

According to an aspect of some embodiments of the present inventionthere is provided an isolated bacteria comprising a nucleic acidsequence encoding a BREX system and a transmissible genetic elementexpressing the BREX system, wherein the isolated bacteria do notendogenously express the BREX system and wherein the BREX systemcomprises brxC/pglY, pglZ and at least one of pglX, pglXL, brxP, brxHI,brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso thatthe BREX system does not comprise pglW, or comprises brxC/pglY, pglZ,pglX, pglW and at least one of brxD and brxHI.

According to some embodiments of the invention, the BREX system is type1 comprising brxA, brxB, brxC/pglY, pglX, pglZ and brxL.

According to some embodiments of the invention

-   -   (i) brxC/pglY is selected from the group of SEQ ID NO: 3-155,        157-765 and 767-1175,    -   (ii) pglZ is selected from the group of SEQ ID NO: 1176-1318,        1320-1856, 1858-2250, 6204 and 6205,    -   (iii) pglX is selected from the group of SEQ ID NO: 2251-3280        and 6186-6201.    -   (iv) pglXI is selected from the group of SEQ ID NO: 3281-3296,        3298-3356 and 3358-3403,    -   (v) brxP is selected from the group of SEQ ID NO: 3404-3440,    -   (vi) brxHI is selected from the group of SEQ ID NO: 3543-3642,    -   (vii) brxHII is selected from the group of SEQ ID NO: 3441-3460,        3462-3511 3513-3542 and 6173-6185,    -   (viii) brxL is selected from the group of SEQ ID NO: 3643-4412,        6165, 6166, 6169, 6170, 6202 and 6203.    -   (ix) brxD is selected from the group of SEQ ID NO: 4413-4488.    -   (x) brxA is selected from the group of SEQ ID NO: 4489-4621,        4623-5086, 5088-5415, 6167, 6168, 6171 and 6172,    -   (xi) brxB is selected from the group of SEQ ID NO: 5416-5947,        6206-6209,    -   (xii) brxF is selected from the group of SEQ ID NO: 5948-5957,        5959-5988 and 5990-6028.    -   (xiii) brxE is selected from the group of SEQ ID NO: 6029-6040,        and    -   (xiv) pglW is selected from the group of SEQ ID NO: 6041-6138.

According to an aspect of some embodiments of the present inventionthere is provided a method of inducing phage sensitivity in a bacterialcell, the method comprising contacting a bacterial cell which expressesa BREX system comprising brxC/pglY, pglZ and at least one of pglX,pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, withthe proviso that the BREX system does not comprise pglW, or comprisingbrxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI; with ananti BREX system agent capable of down regulating a BREX gene selectedfrom the group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI,brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, thereby inducingsensitivity of the bacterial cell to phage infection.

According to some embodiments of the invention, the contacting iseffected ex-vivo or in-vitro.

According to some embodiments of the invention, the contacting iseffected in-vivo.

According to some embodiments of the invention, there is provided anisolated bacteria generated according to the method.

According to some embodiments of the invention, there is provided amethod for preparing a food, food additive, feed, nutritionalsupplement, probiotic supplement, a personal care product, a health careproduct, and a veterinary product comprising adding to the food, foodadditive, feed, nutritional supplement, probiotic supplement, a personalcare product, a health care product, and a veterinary product theisolated polynucleotide, the construct, the composition, the isolatedcell or the bacteria, thereby preparing the food, food additive, feed,nutritional supplement, probiotic supplement, personal care product,health care product, and veterinary product.

According to an aspect of some embodiments of the present inventionthere is provided a method for preparing a food, food additive, feed,nutritional supplement, probiotic supplement, a personal care product, ahealth care product, and a veterinary product comprising adding to thefood, food additive, feed, nutritional supplement, probiotic supplement,a personal care product, a health care product, and a veterinary producta bacteria which expresses on a transmissible genetic element a BREXsystem comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP,brxHI, brxHII, brxL brxD, brxA, brxB, brxF, and brxE, with the provisothat the BREX system does not comprise pglW, or comprising brxC/pglY,pglZ, pglX, pglW and at least one of brxD and brxHI, thereby preparingthe food, food additive, feed, nutritional supplement, probioticsupplement, personal care product, health care product, and veterinaryproduct.

According to some embodiments of the invention, the transmissiblegenetic element comprises a conjugative genetic element or mobilizablegenetic element.

According to some embodiments of the invention, the food or feed is adairy product.

According to some embodiments of the invention, the cell is a bacteria.

According to some embodiments of the invention, the bacteria is aspecies selected from the group consisting of Escherichia, Shigella,Salmonella, Erwinia, Yersinia, Bacillus, Vibrio, Legionella,Pseudomonas, Neisseria, Bordetella, Helicobacter, Listeria,Agrobacterium, Staphylococcus, Streptococcus, Enterococcus, Clostridium,Corynebacterium, Mycobacterium, Treponema, Borrelia, Francisella,Brucella, Campylobacter, Klebsiella, Frankia, Bartonella, Rickettsia,Shewanella, Serratia, Enterobacter, Proteus, Providencia, Brochothrix,and Brevibacterium.

According to some embodiments of the invention, the bacteria is a lacticacid bacteria.

According to some embodiments of the invention, the bacteria is aspecies selected from the group consisting of Lactococcus species,Streptococcus species, Lactobacillus species, Leuconostoc species,Oenococcus species, Pediococcus species, Bifidobacterium, andPropionibacterium species.

According to some embodiments of the invention, there is provided afood, food additive, feed, nutritional supplement, probiotic supplement,a personal care product, a health care product, and a veterinary productcomprising the isolated polynucleotide, the construct, the composition,the isolated cell or the isolated bacteria.

According to some embodiments of the invention, the product furthercomprises a dairy product.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a microbial infection in asubject in need thereof, the method comprising contacting the bacteriawith an anti BREX system agent capable of down regulating a BREX geneselected from the group consisting of brxC/pglY, pglZ, pglX, pglXI,brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW,thereby treating the infection.

According to some embodiments of the invention, the method furthercomprising administering to the subject a phage therapy.

According to some embodiments of the invention, the method furthercomprising administering to the subject an antibiotic.

According to some embodiments of the invention, the method furthercomprising administering to the subject a phage therapy and/or anantibiotic.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for killing abacteria comprising a packaging material packaging an anti BREX systemagent capable of down regulating a BREX gene selected from the groupconsisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL,brxD, brxA, brxB, brxF, brxE, and pglW, and a phage.

According to an aspect of some embodiments of the present inventionthere is provided an anti-microbial composition comprising as activeingredient an anti BREX system agent capable of down regulating a BREXgene selected from the group consisting of brxC/pglY, pglZ, pglX, pglXI,brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, andan acceptable carrier or diluent.

According to some embodiments of the invention, the composition furthercomprising a phage.

According to some embodiments of the invention, the anti BREX systemagent is administered in a formulation suitable for cell penetration.

According to some embodiments of the invention, the anti BREX systemagent is selected from the group consisting of a nucleic acid suitablefor silencing expression, aptamers, small molecules and inhibitorypeptides.

According to some embodiments of the invention, the anti BREX systemagent is directed against pglX.

According to some embodiments of the invention, the anti BREX systemagent is directed against brxC/pglY or pglZ.

According to an aspect of some embodiments of the present inventionthere is provided a method of screening for identifying phage useful forinfecting a bacteria, the method comprising:

(a) contacting a phage with a bacteria expressing BREX system comprisingbrxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII,brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREXsystem does not comprise pglW or comprising pglY, brxC/pglZ, pglX, pglWand at least one of brxD and brxHI;

(b) monitoring phage sensitivity of the bacteria, wherein an increase inphage sensitivity of the bacteria in the presence of the phage comparedto phage sensitivity in the absence of the phage is indicative of aphage useful for infecting the bacteria.

According to some embodiments of the invention, the carrier is apharmaceutically acceptable carrier.

According to some embodiments of the invention, the BREX system ischaracterized by at least one of

(i) not being an abortive infection system;

(ii) not being a restriction modification system;

(iii) not preventing phage adsorption to a bacteria expressing same.

According to some embodiments of the invention, the pglX is a methylase.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-C demonstrate pglZ phylogeny and the classification ofBacteriophage Exclusion (BREX) subtypes. FIG. 1A is a phylogenetic treerepresenting pglZ protein occurrences. The tree is color coded accordingto the different BREX subtypes. Gene order and genomic organization ofeach BREX subtype is illustrated next to its relevant branch on the pglZtree. Numbers depict bootstap values. FIG. 1B is a pie chartdemonstrating the prevalence of the different BREX subtypes within theBREX superfamily system among the sequenced genomes that were analyzed.FIG. 1C shows representative appearances of the pglZ gene in a type 1BREX six-gene cluster.

FIG. 2 shows local alignment between brxC from Bacillus cereus H3081.97(type 1 BREX) and pglY from Streptomyces coelicolor A3(2) (type 2 BREX)demonstrating that the alignment between the two genes spans the P-loopdomain [GXXXXGK(T/S), DUF2791, SEQ ID NO: 6162)]. Alignment coverage:4%, e-value: 8e-09, identity: 24/57 (42%). Locus tags marked inparentheses.

FIG. 3 shows the structural alignment between brxA from Magnetospirillummagneticum (PDB entry 3BHW) and NusB from Aquifex aelicus (PDB entry3R2C) demonstrating structural homology between brxA and NusB. BrxA ismarked in red; NusB is marked in blue; NusE is marked in cyan; RNA ismarked in grey. Alignment length: 44 amino acids.

FIGS. 4A-M demonstrate that integration of type 1 BREX system fromBacillus cereus H3081.97 into Bacillus subtilis BEST7003 strain confersresistance of the latter to phage infection. FIG. 4A depicts the type 1BREX locus in Bacillus cereus H3081.97. Coordinates below the genesdenote the position along the NZ_ABDLO1000007 contig in the draft genomeof Bacillus cereus H3081.97. The orange box within brxC represents theposition of the ATPase p-loop motif. FIG. 4B depicts the operonorganization of the Bacillus cereus BREX system integrated in theBacillus subtilis genome. FIGS. 4C-F are bivariate graphs indicating 12hours culture dynamics of the control strain (BREX⁻, black) versus type1 BREX-containing (red) strain of Bacillus subtilis following infectionwith different phages, as evaluated by optical density measurements in a96-well plate format. Bacterial strains were exposed to phage at Time=0.Each graph represents 3 experiments with three technical triplicates foreach biological replicate. Error bars represent SEM. FIG. 4C illustratesnon-infected control (BREX⁻) and type 1 BREX-containing Bacillussubtilis cultures. FIGS. 4D-M illustrate culture dynamics of the BREX⁻and type 1 BREX-containing Bacillus subtilis cultures followinginfection with Φ3T (FIG. 4D), rho10 (FIG. 4E), SP82G (FIG. 4F), SPO1(FIG. 4G), 0105 (FIG. 4H). SPO2 (FIG. 4I), SP16 (FIG. 4J), Zeta (FIG.4K), SPβ (FIG. 4L), and rho14 (FIG. 4M) phages.

FIGS. 5A-B are bivariate graphs demonstrating culture dynamics in thecontrol strain (BREX⁻, black) versus type 1 BREX-containing (red) strainof Bacillus subtilis BEST7003 cultures following infection withdifferent phages, as evaluated by optical density measurements. FIGS.5A-B depict culture dynamics over an extended period (>30 hours)following infection with SOP1 (FIG. 5A) and SP28G (FIG. 5B)demonstrating temporally reproducible culture decline of the controlBREX strain, but irreproducible culture decline at stochastic timepoints of the type 1 BREX⁺ strain. Re-growth following culture crashrepresents phage-resistant mutants. Each curve in the graphs representsa single technical replicate.

FIG. 6 is a bivariate graph demonstrating phage production during aone-step phage growth curve experiment with control strain (BREX⁻,black) versus type 1 BREX-containing (red) strain of Bacillus subtilisBEST7003 following infection with SPO1. The Y-axis represents absolutephage concentrations; the time points of maximal burst for BREX-lackingand BREX-containing strains are marked with black and red arrows,respectively. Error bars represent standard deviation of biologicaltriplicates.

FIG. 7 demonstrates that BREX system confers resistance to phage firstcycle of infection. Shown is a bivariate graph of phage productionduring a one-step phage growth curve experiment with control strain(BREX⁻, black) versus type 1 BREX-containing (red) strain of Bacillussubtilis BEST7003 infected with Φ3T. The Y-axis represents relativephage concentrations normalized to the value at the beginning ofinfection. Error bars represent standard deviation of biologicaltriplicates.

FIG. 8 is a representative PCR photograph showing the presence oflysogens during a phage infection time course in the control Bacillussubtilis strain (BREX⁻, black), but not in type 1 BREX-containingBacillus subtilis strain (BREX⁺, red), or uninfected (U) strains.Amplicons for the bacterial DNA, phage DNA, and lysogen-specific DNA are293 bp, 485 bp, and 1218 bp, respectively.

FIG. 9 is a bivariate graph demonstrating culture dynamics in thecontrol strain (BREX⁻, black) versus type 1 BREX-containing (red) strainof Bacillus subtilis BEST7003 cultures following infection withincreasing MOI of Φ3T phage showing that increasing the MOI shortens thetime to culture crash for the BREX⁻ strain, but minimally influences thegrowth of the BREX⁺ strains. Error bars represent standard deviation oftechnical triplicates.

FIG. 10 is a bar graph representing the number of extracellular phagesin the control strain (BREX⁻, black) versus type 1 BREX-containing (red)strain of Bacillus subtilis BEST7003 cultures 15 minutes followinginfection with phage Φ3T.

FIG. 11 is a bivariate graph illustrating that phage DNA replication isobserved in control Bacillus subtilis BEST7003 strain (BREX, black) butdoes not occur in type 1 BREX-containing strain of Bacillus subtilisBEST7003 (BREX⁺, red). The Y-axis represents relative phage DNAconcentration normalized to the value at the beginning of infection, asevaluated by Illumina sequencing of total cellular DNA.

FIG. 12 is a southern blot photograph demonstrating phage Φ3T genome atdifferent time points following infection of control (BREX⁻, black) vs.type 1 BREX-containing (BREX⁺, red) Bacillus subtilis BEST7003 strains.Numbers marking each lane indicate the time (in minutes) followinginfection. U lanes indicate uninfected control.

FIGS. 13A-B demonstrate the methylation activity of BREX. FIG. 13Adepicts the consensus sequence around the modified base in the type 1BREX-containing Bacillus subtilis BEST7003 strain. The arrow marks themodified base. FIG. 13B is a table summarizing the statistics of themodified motifs in the type 1 BREX-containing Bacillus subtilis BEST7003strain.

FIGS. 14A-B are bivariate graphs demonstrating culture dynamics ofnon-infected (FIG. 14A) or following infection with phage Φ3T (FIG. 14B)in the control strain (BREX⁻, black), type 1 BREX-containing (red) andtype 1 BREX without pglX-containing (pglXΔ, green) strains of Bacillussubtilis BEST7003 cultures, as evaluated by optical densitymeasurements.

FIG. 15 is a Common Tree of bacteria and archaea as represented in theNCBI Taxonomy resource demonstrating the Distribution of BREX systemsacross the phylogenetic tree of bacteria and archaea. Organisms in whicha BREX system exists are colored following the BREX subtype color codefrom FIG. 1A. Extensive horizontal transfer is observed by the lack ofcoherence between the species tree and the pglZ phylogeny.

FIG. 16 is a phylogenetic tree representing brxC/pglY proteinoccurrences as determined by aligning only the P-loop domain showingthat the brxC/pglY phylogeny follows the classification of BREX subtypesand co-evolves with pglZ. The tree is color coded according to thedifferent BREX subtypes as in FIG. 1A. Only brxC/PglY proteins appearingin complete systems were taken for this analysis.

FIGS. 17A-C illustrate frequent irregularities in the adenine-specificmethylase pglX in BREX type 1. FIG. 17A depicts irregular genotypes(duplication, inversion and premature stop codon) associated with pglX.FIG. 17B depicts genomic organization of BREX system type 1 inMethanobrevibacter smithii ATCC 35061 (NC_009515 SEQ ID NO: 6163). FIG.17C depicts genomic organization of BREX system type 1 in Lactobacillusrhamnosus GG. A cassette switch between the short and the long forms ofpglX is observed when the sequences of two isolates of Lactobacillusrhamnosus GG (accessions FM179322 (NC_013198 SEQ ID NO: 1) and AP011548(NC_017482 SEQ ID NO: 2) respectively are compared. Repeat sequencesbetween the short and long forms are in black.

FIG. 18 is an agarose gel photograph of DNA extracted from type 1BREX-containing and BREX-lacking strains of Bacillus subtilis BEST7003cultures, following time course infection with phage Φ3T demonstratingno degradation of host DNA.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates topolynucleotides encoding BREX system polypeptides and methods of usingsame.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

The continuous bacteria-phages arms race has led to rapid evolution ofboth anti-phage bacterial resistance systems and counter-resistancemechanisms developed by phages, many of which are yet uncharacterized. Abroad array of food products, commodity chemicals, and biotechnologyproducts are manufactured industrially by large-scale bacterialfermentation of various substrates. Development of defense strategiesand systems to curtail the propagation and evolution of phages infermentation vats is therefore warranted. On the counter arm, properlyformulated and applied phages have sufficient potential to curebacterial infections addressing the therapeutic need for newantibiotics.

The PGL system has previously been reported as conferring phageresistance manifested by attenuation of phage growth in the secondcycle.

Whilst reducing the present invention to practice, the present inventorshave now uncovered a novel multi-gene phage resistance system broadlydistributed in bacteria and archaea, which the present inventors denotedBREX (Bacteriophage Elusion) system. The newly discovered BREX systemshares some structural and functional similarities with the previouslydescribed PGL system. The abundance of this system and the efficiency inwhich it protects against phages implies that it plays an important roleas a major line of defense encoded by bacteria against phages.

Specifically, the present inventors have uncovered that BREX systemconfers complete or partial resistance against phages spanning a widephylogeny of phage types, including lytic and temperate (also referredlysogenic) phages, even in the first cycle of infection. Alternatively,mutations (e.g.; frame shift in pglX) affecting the functionality of theBREX system abrogate phage resistance.

Taken together, the present teachings suggest that BREX system andfunctional portions thereof can be used for conferring phage resistance.Such naturally and engineered bacteria can be utilized for example inthe dairy industry, where phages cause serious annual losses, as well asin other industries that rely on large-scale bacterial fermentation forbiotechnological production. Alternatively, anti-BREX system agents canbe used as antibiotics.

As is illustrated hereinunder and in the examples section which follows,the present inventors have uncovered that BREX system confers completeor partial resistance against phages spanning a wide phylogeny of phagetypes, including lytic and temperate phages, even in the first cycle ofinfection. Even more so, mutations (e.g.; frame shift in pglX) affectingthe functionality of the BREX system abrogate phage resistance.Specifically, the present inventors have shown that the BREX systemexists in almost 10% of sequenced microbial genomes, and can be dividedinto six coherent subtypes containing 4-8 genes each, two of which arecore genes, pglZ and brxC/pglY, present in all subtypes (Examples 1-2,Tables 1-8, FIGS. 1A-C). As detailed in Example 2 and Tables 2-7, inmost cases, a single BREX system per organism was found, and in severalof the identified systems, one of the genes was either missing or hasbecome a pseudogene. The inventors have further demonstrated that theBREX system undergoes extensive horizontal transfer, with subtype 1, themost frequent subtype of this system, possibly the ancestral form ofBREX (Example 5, FIGS. 1A-C, 16.)

The inventors have further demonstrated (Examples 3 and 4, FIGS. 4A-M,5A-B, 6, 7 and Table 9) that integration of the BREX type 1 system intoBacillus subtilis strain lacking an endogenous BREX system conferscomplete protection from infection by the temperate SPβ, SP16, Zeta,Φ3T, and SPO2 phages and partial protection from infection by the lyticSP82G and SPO1 phages, even in the first cycle of infection.

The present inventors have gained insight into BREX mechanism of action(Example 4, FIGS. 8-12), accordingly it is demonstrated that integrationof the type 1 BREX system into Bacillus subtilis strain allows phageadsorption but prevents phage lysogeny and phage DNA replication. Inaddition, the system methylates the host chromosomal DNA at a specificmotif while sparing the phage DNA and this methylation is likely to beessential for the system's activity. Specifically, the methylase genepglX was essential for the function of the type 1 BREX system andpresented high rates of irregularities (Example 4. FIGS. 13A-B and 14A-Band Example 6. FIGS. 17A-B), thus marking pglX as possibly undergoingfrequent phase-variation, and suggesting that this gene might conferspecificity in the BREX system, or, alternatively, is particularlytoxic. The data strongly indicates that the system does not an act viathe previously described PGL mechanism or via abortive infection (Abi)or simple restriction/modification mechanisms.

Consequently, the present invention provides methods and compositionsfor use in the food, feed, medical and veterinary industries to conferphage resistance. On the other hand, the present invention providesmethods suitable for use in the food, feed, medical and veterinaryindustries to generate phage with broader host range that can be usedfor more effective bio-control of bacteria.

Thus, according to a first aspect of the present invention, there isprovided an isolated polynucleotide encoding a BREX system comprising anucleic acid sequence encoding the BREX system comprising brxC/pglY,pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD,brxA, brxB, brxF, and brxE, with the proviso that the BREX system doesnot comprise pglW, and wherein the BREX system confers phage resistanceto a bacteria recombinantly expressing same.

According to a second aspect of the present invention, there is providedan isolated polynucleotide encoding a BREX system comprising a nucleicacid sequence encoding the BREX system comprising brxC/pglY, pglZ, pglX,pglW and at least one of brxD and brxHI, and wherein the BREX systemconfers phage resistance to a bacteria recombinantly expressing same.

According to another aspect of the present invention, there is providedan isolated polynucleotide comprising a nucleic acid sequence encoding aBREX system comprising:

(i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL;

(ii) brxA, brxB, (brxC/pglY)_(x2), pglX, pglZ and brxHII;

(iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI;

(iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA;

(v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHII; or

(vi) brxP, brxC/pglY, pglZ and brxL

As used herein the term “isolated” refers to at least partiallyseparated from the natural environment, physiological environment e.g.,a microorganism e.g., bacteria.

As used herein “BREX system” (previously denoted “PYZA system”) or a“functional BREX system”, refers to a multi-gene system which comprisesBRX and/or PGL genes which expression confers phage resistance.

According to specific embodiments, the BREX system is characterized byat least one of

(i) not being an abortive infection system;

(ii) not being a restriction modification system;

(iii) not preventing phage adsorption to a bacteria expressing same.

The BREX system may be characterized by one, two or all i.e.: (i); (ii);(iii); (i)+(ii); (i)+(iii); (ii)+(iii) and (i)+(ii)+(iii).

According to a specific embodiment the BREX system is characterized by(i)+(ii)+(iii).

As used herein “abortive infection (Abi) system” refers to a controlledcell death of an infected bacterial cell which takes place prior to theproduction of phage progeny, thus protecting the culture from phagepropagation. Methods of analyzing Abi include, but are not limited tocell survival assays using high multiplicity of infection, one stepgrowth assays and determination of phage DNA replication by e.g. DNAsequencing and southern blot analysis as further described hereinbelow.

As used herein “restriction modification system” refers to therecognition and cleavage of foreign DNA. Typically, a restrictionmodification system comprises a restriction enzyme having an activity ofcleaving DNA and a modification enzyme capable of protecting host DNAfrom the cleavage by the restriction enzyme e.g. by methylating the hostDNA. Analyzing restriction modification mode of action include, but isnot limited to, evaluation of host specific methylation, presence ofdegraded foreign DNA and host cell death in the absence of themodification enzyme by methods described infra.

As used herein “adsorption” refers to the attachment to the host (e.g.bacteria) cell surface via plasma membrane proteins and glycoproteins.Methods of analyzing phage adsorption include, but are not limited toenumerating free phages in bacterial cultures infected with the phagesimmediately after phage addition and at early time points (e.g. 30minutes) following phage addition as further described hereinbelow.

As used herein “phage resistance” refers to a phage infection resistancewhich can be a first or a second cycle resistance. The phage can be alytic phage or a temperate (lysogenic) phage. According to a specificembodiment the BREX system confers phage resistance to a first cyclephage infection. According to yet other specific embodiments, BREXsystem confers resistance to lytic phages.

According to a specific embodiment, BREX system confers resistance tophage lysogeny.

As used herein, the term “lysogeny” refers to the incorporation of thephage genetic material inside the genome of the host (e.g. bacteria).Methods of analyzing phage lysogent are well known in the art andinclude, but not limited to, DNA sequencing and PCR analysis.

According to another specific embodiment, BREX system confers resistanceto phage DNA replication.

According to specific embodiments. BREX system does not conferresistance to phages Φ105, rho10 and rho14.

As used herein, “phage resistance” refers to an increase of at least 10%in bacterial resistance towards a phage in comparison to bacteria of thesame species under the same developmental stage (culture state) whichdoes not express a BREX system, as may be manifested in e.g. bacterialviability, phage lysogeny and phage DNA replication. According to aspecific embodiment, the increase is in at least 20%, 30%, 40% or evenhigher say, 50%, 60%, 70%, 80%, 90% or more than 100%.

Assays for testing phage resistance are well known in the art andmentioned hereinbelow.

According to specific embodiments, BREX system confers resistance to aplasmid. The plasmid may undergo integration into the bacterial genomeor may be episomal.

According to a specific embodiment, the plasmid is episomal.

As used herein, “plasmid resistance” refers to an increase of at least5% in bacterial resistance towards a plasmid in comparison to bacteriaof the same species under the same developmental stage (culture state)which does not express a BREX system, as may be manifested in e.g.viability. According to a specific embodiment, the increase is in atleast 10%, 20%, 30%, 40% or even higher say, 50%, 60%, 70%, 80%, 90% ormore than 100%.

Assays for testing plasmid resistance are well known in the art andinclude, but not limited to, a transformation assay such as described inItaya and Tsuge [Methods Enzymol (2011) 498:427-47].

As used herein, “expressing” or “expression” refers to gene expressionat the RNA and/or protein level.

As used herein the “Phage growth Limitation” abbreviated as PGL refersto a cluster of genes which were previously described in Streptomycescoelicolor A3(2) (Chinenova T. A. et al, 1982; Sumby, P. & Smith, M. C.2002, herein incorporated by reference in its entirety).

As used herein the “Bacteriophage Exclusion” abbreviated as BREX refersto a cluster of genes some of which were previously described inStreptomyces coelicolor A3(2) (Chinenova T. A. et al, 1982; Sumby, P. &Smith, M. C. 2002,).

According to specific embodiments, the BREX genes which compose the BREXsystem comprise brxC/pglY, pglZ pglW, pglX, pglXI, brxP, brxHI, brxHII,brxL, brxD, brxA, brxB, brxF, and brxE, which can be divided into sixcoherent subtypes comprising 4-8 genes each, in which the gene order andcomposition is conserved.

Thus, the BREX subtypes according to some embodiments of the presentinvention are selected from the group consisting of:

(1) brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI,brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso thatthe functional BREX system does not comprise pglW.

(2) brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI,

(3i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL (also may be referred toas Type 1).

(3ii) brxA, brxB, (brxC/pglY)_(x2), pglX, pglZ and brxHII (also may bereferred to as Type 5),

(3iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI (also maybe referred to as Type 6),

(3iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA (also may bereferred to as Type 3),

(3v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHI (also may be referredto as Type 2), or

(3vi) brxP, brxC/pglY, pglZ and brxL (also may be referred to as Type4).

Thus, specific examples of BREX systems which can be used according tothe present teachings include but are not limited to BREX system type 1,BREX system type 2, BREX system type 3. BREX system type 4, BREX systemtype 5 and BREX system type 6 (see FIG. 1A).

According to specific embodiments, BREX system type 1 (previouslydenoted PYZA system type 1a) comprises brxA, brxB, brxC/pglY, pglX, pglZand brxL; BREX system type 5 (previously denoted PYZA system type 1b)comprises brxA, brxB, (brxC/pglY)_(x2), pglX, pglZ and brxHII; BREXsystem type 6 (previously denoted PYZA system type 1c) comprises brxE,brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI; BREX system type 3(previously denoted PYZA system type 2) comprises brxF, brxC/pglY,pglXI, brxHII, pglZ and brxA; BREX system type 2 (previously denotedPYZA system type 3) comprises pglW, pglX, brxC/pglY, pglZ, brxD andbrxHI; and BREX system type 4 (previously denoted PYZA system type 4)comprises brxP, brxC/pglY, pglZ and brxL.

According to specific embodiments the BREX system is type 1 comprisingbrxA, brxB, brxC/pglY, pglX, pglZ and brxL.

Two of the six genes found in type 1 BREX conserved cluster sharehomology with genes from the previously reported PGL system^(10,11):pglZ, coding for a protein with a predicted alkaline phosphatase domain,and pglX, coding for a protein with a putative methylase domain. Thefour additional genes include (i) a Ion-like protease-domain gene,denoted herein as brxL; (ii) brxA; (iii) brxB; and (iv) a ˜1200 aminoacid protein with an ATP binding motif (GXXXXGK[T/S]), denoted herein asbrxC. The preferential localization of this conserved gene cluster inthe genomic vicinity of other defense genes suggests that it is a novelphage defense system.

The phage defense system originally described in Streptomyces coelicolorA3(2) as PGL is defined according to the present teachings as a type 2BREX. While the PGL was described to be composed of four genes, pglW,pglX, pglY and pglZ, the present teaching suggest that 2 more genes,brxD and brxHI, are an integral part of the type 2 BREX system. Inaddition, pglW, an integral part of the previously described PGL, existsexclusively in type 2 BREX subtype.

The major phage resistance systems that were characterized to date,including the restriction-modification and CRISPR-Cas systems, encodemostly for proteins that interact with and manipulate DNA and RNAmolecules. While the BREX system contains such proteins includingmethylases and helicases it also contains genes coding for proteinspredicted to be involved in the manipulation of other proteins, such asthe Ion-like protease, brxL, the predicted alkaline phosphatase, pglZ,and the serine/threonine kinase, brxW. Thus, according to specificembodiments, the defense mechanism employed by the BREX system takesplace later in the infection where phage proteins are already producedand can be manipulated by pglZ and/or brxL.

According to other specific embodiments, BREX proteins target phageproteins co-injected with the phage DNA early in the infection cycle.

According to specific embodiments the BREX system acts before phage DNAreplication.

According to specific embodiments, BREX proteins interact with otherbacterial-encoded proteins to regulate BREX activity.

As used herein, the terms “pglY”, “brxC” and “brxC/pglY” refer to thepolynucleotide and expression product e.g., polypeptide of the PGLY orBRXC gene. The polypeptide product of the PGLY and BRXC genes typicallycontains p-loop ATPase/ATP binding domain DUF2791 (pfam10923, SEQ ID NO:6162) and a DUF499 domain. brxC/pglY together with pglZ and at least oneof pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE,and pglW, comprise a BREX system.

According to specific embodiments, brxC/pglY is selected from the groupconsisting of SEQ ID NO: 3155, 157-765 and 767-1175.

As used herein, the term “pglZ” refers to the polynucleotide andexpression product e.g., polypeptide of the PGLZ gene. The polypeptideproduct of the PGLZ gene typically contains an alkaline phosphatasedomain pfam08665. pglZ together with brxC/pglY and at least one of pglX,pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, andpglW, comprise a BREX system.

According to specific embodiments pglZ is selected from the groupconsisting of SEQ ID NO: 1176-1318, 1320-1856, 1858-2250, 6205 and 6204.

As used herein, the term “pglX” refers to the polynucleotide andexpression product e.g., polypeptide of the PGLX gene. The polypeptideproduct of the PGLX gene typically contains an adenine-specific DNAmethyltransferase domain pfam13659 (COG1002/COG0286). pglX together withat least brxC/pglY and pglZ and optionally at least one of pglXI, brxP,brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE and pglW, comprise aBREX system. pglX is a critical gene as the present inventors have shownthat it presented high rates of irregularities in the BREX systemsdocumented and a frame shift mutation in this gene in one of the type 1BREX-containing Bacillus subtilis strains obtained was not activeagainst any of the tested phages. In addition, Bacillus subtilis strainscontaining a type 1 BREX having a deletion of pglX were sensitive to allthe phages tested.

According to specific embodiments pglX is selected from the groupconsisting of SEQ ID NO: 2251-3280 and 6186-6201.

According to specific embodiments, pglX is a methylase.

According to a specific embodiment, the pglX methylase and pglXImethylase are analogous in BREX systems types 1 and 3, respectively.

According to specific embodiments, the methylase of the BREX systemmethylates the bacterial DNA.

According to a specific embodiment, the methylase of the BREX systemdrives motif-specific (e.g. an adenine residue in TAGGAG motif)methylation on the genomic DNA of a bacteria expressing same. Accordingto specific embodiments the methylation is non-polindromic. According tospecific embodiments the BREX system methylase does not methylate aphage genome.

According to specific embodiments the methylation serves as part of theself/non-self recognition machinery of BREX.

According to a specific embodiment, type 4 BREX does not contain amethylase.

According to a specific embodiment the pglX methylase and brxP reductaseare analogous in BREX systems types 1 and 4, respectively.

Methods of assessing DNA methylation and, more specifically,adenine-specific methylation are well known in the art and include e.g.the PacBio sequencing platform [Murray et al. Nucleic acids research(2012) 40: 11450-11462].

As used herein, the term “pglXI” refers to the polynucleotide andexpression product e.g., polypeptide of the PGLXI gene. The polypeptideproduct of the PGLXI gene typically contains an adenine-specific DNAmethylase COG0863/COG1743 (pfam 01555). pglXI together with at leastbrxC/pglY and pglZ and optionally at least one of pglX, brxP, brxHI,brxHII, brxL, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREXsystem.

According to specific embodiments pglXI is selected from the groupconsisting of SEQ ID NO: 3281-3296, 3298-3356 and 3358-3403.

As used herein, the term “brxP” (previously denoted “pglPA”) refers tothe polynucleotide and expression product e.g., polypeptide of the BRXPgene. The polypeptide product of the BRXP gene typically contains aphosphoadenosine phosphosulfate reductase domain COG0175 (pfam01507),and a pfam13182 domain. brxP together with at least brxC/pglY and pglZand optionally at least one of pglX, pglXI, brxHI, brxHII, brxL, brxD,brxA, brxB, brxF, brxE and pglW, comprise a BREX system.

According to specific embodiments brxP is selected from the groupconsisting of SEQ ID NO: 3404-3440.

As used herein, the term “brxHI” (previously denoted “pglHI”) refers tothe polynucleotide and expression product e.g., polypeptide of the BRXHIgene. The polypeptide product of the BRXHI gene typically contains anLhr-like a helicase domain COG1201. brxHI together with at leastbrxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP,brxHII, brxL, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREXsystem.

According to specific embodiments brxHI is selected from the groupconsisting of SEQ ID NO: 3543-3642.

As used herein, the term “brxHII” (previously denoted “pglHII”) refersto the polynucleotide and expression product e.g., polypeptide of theBRXHII gene. The polypeptide product of the BRXHII gene typicallycontains a DNA/RNA helicase domain COG0553. brxHII together with atleast brxC/pglY and pglZ and optionally at least one of pglX, pglXI,brxP, brxHI, brxL, brxD, brxA, brxB, brxF, brxE and pglW, comprise aBREX system.

According to specific embodiments brxHII is selected from the groupconsisting of SEQ ID NO: 3441-3460, 3462-3511, 3513-3542 and 6173-6185.

As used herein, the term “brxL” (previously denoted “pglL”) refers tothe polynucleotide and expression product e.g., polypeptide of the BRXLgene. The polypeptide product of the BRXL gene typically contains aion-like protease domain COG4930. brxL together with at least brxC/pglYand pglZ and optionally at least one of pglX, pglXI, brxP, brxHI,brxHII, brxD, brxA, brxB, brxF, brxE and pglW, comprise a BREX system.

According to specific embodiments brxL is selected from the groupconsisting of SEQ ID NO: 3643-4412, 6165, 6166, 6169, 6170, 6202 and6203.

As used herein, the term “brxD” (previously denoted “pglD”) refers tothe polynucleotide and expression product e.g., polypeptide of the BRXDgene. The polypeptide product of the BRXD gene typically contains an ATPbinding domain DUF2791 (pfam10923). brxD together with at leastbrxC/pglY and pglZ and optionally at least one of pglX, pglXI, brxP,brxHI, brxHII, brxL brxA, brxB, brxF, brxE and pglW, comprise a BREXsystem.

According to specific embodiments brxD is selected from the group ofconsisting SEQ ID NO: 4413-4488.

As used herein, the term “brxA” (previously denoted “pglA”) refers tothe polynucleotide and expression product e.g., polypeptide of the BRXAgene. The polypeptide product of the BRXA gene typically contains aDUF1819 (pfam08849) domain. The brxA protein displays significantstructural homology to NusB spanning the RNA-binding interface, as wellas part of the protein:protein interaction interface of NusB with NusE.In light of this similarity, according to specific embodiments brxA isan RNA binding protein. According to specific embodiments, brxA has arole in interfering with the phage infection cycle by disruptinganti-termination events essential for the phage cycle. brxA togetherwith at least brxC/pglY and pglZ and optionally at least one of pglX,pglXI, brxP, brxHI, brxHII, brxL, brxD, brxB, brxF, brxE and pglWcomprise a BREX system.

According to specific embodiments brxA is selected from the groupconsisting of SEQ ID NO: 4489-4621, 4623-5086, 5088-5415, 6167, 6168,6171 and 6172.

As used herein, the term “brxB” (previously denoted “pglB”) refers tothe polynucleotide and expression product e.g., polypeptide of the BRXBgene. The polypeptide product of the BRXB gene typically contains aDUF1788 (pfam08747) domain. brxB together with at least brxC/pglY andpglZ and optionally at least one of pglX, pglXI, brxP. brxHI, brxHII,brxL, brxD, brxA, brxF, brxE and pglW, comprise a BREX system.

According to specific embodiments brxB is selected from the groupconsisting of SEQ ID NO: 5416-5947 and 6206-6209.

As used herein, the term “brxF” (previously denoted “pglC”) refers tothe polynucleotide and expression product e.g., polypeptide of the BRXFgene. The polypeptide product of the BRXF gene typically contains anATPase domain, brxF together with at least brxC/pglY and pglZ andoptionally at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD,brxA, brxB, brxE and pglW, comprise a BREX system.

According to specific embodiments brxF is selected from the groupconsisting of SEQ ID NO: 5948-5957, 5959-5988 and 5990-6028.

As used herein, the term “brxE” (previously denoted “pglE”) refers tothe polynucleotide and expression product e.g., polypeptide of the BRXEgene. brxE together with at least brxC/pglY and pglZ and optionally atleast one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB,brxF, and pglW, comprise a BREX system.

According to specific embodiments brxE is selected from the groupconsisting of SEQ ID NO: 6029-6040.

As used herein, the term “pglW” refers to the polynucleotide andexpression product e.g., polypeptide of the PGLW gene. The polypeptideproduct of the PGLW gene typically contains a serine/threonine kinasedomain COG0515. pglW together with brxC/pglY, pglZ and pglX, and atleast one of brxD and brxHI, and optionally at least one of pglXI, brxP,brxHII, brxL, brxA, brxB, brxF, and brxE, comprise a BREX system.

According to specific embodiments pglW is selected from the groupconsisting of SEQ ID NO: 6041-6138.

The terms “brxC/pglY”, “pglZ”, “pglX”, “pglXI”, “brxP”, “brxHI”,“brxHII”, “brxL”, “brxD”, “brxA”, “brxB”, “brxF”, “brxE”, and “pglW”also refers to functional brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI,brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW homologues whichexhibit the desired activity (i.e., conferring phage resistance). Suchhomologues can be, for example, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100% identical or homologous to thepolypeptide SEQ ID NO: 614-765, 767-1175, 1714-1856, 1858-2250, 6204,2766-3280, 6186, 6188, 6190, 6192, 6194, 6196, 6198, 6200, 3343-3356,3358-3403, 3422-3440, 3492-3511, 3513-3542, 6173, 6175, 6178, 6180,6182, 6184, 3593-3642, 4028-4412, 6165, 6169, 6202, 4438-4488,4953-5086, 5088-5415, 6167, 6171, 5570-5947, 6206, 6208, 5979-5988,5990-6028, 6035-6040 and 6090-6138, respectively, or 80%, at least 81%,at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% identical to thepolynucleotide sequence encoding same (as further describedhereinbelow). The homolog may also refer to an ortholog, a deletion,insertion, or substitution variant, including an amino acidsubstitution.

Alternatively or additionally, homology can be based on shared motifs[e.g., the p-loop motif GXXXXGK(T/S) (DUF2791, SEQ ID NO: 6162) andDUF499 motifs present in pglY] combined with the conserved size of thegene in the different subtypes and the location of the gene in the genecluster.

Sequence identity or homology can be determined using any protein ornucleic acid sequence alignment algorithm such as Blast, ClustalW,MUSCLE, and HHpred.

As used herein the term “polynucleotide” refers to a single or doublestranded nucleic acid sequence which is isolated and provided in theform of an RNA sequence, a complementary polynucleotide sequence (cDNA),a genomic polynucleotide sequence and/or a composite polynucleotidesequences (e.g., a combination of the above).

According to specific embodiments the polynucleotides of the presentinvention are part of a nucleic acid construct comprising thepolynucleotide encoding the BREX system and at least one cis-actingregulatory element for directing expression of the nucleic acidsequence.

Teachings of the invention further contemplate that the polynucleotidesare part of a nucleic acid construct system where the BREX genes areexpressed from a plurality of constructs.

Thus, the present invention further provides for a nucleic acidconstruct system comprising at least two nucleic acid constructsexpressing a BREX system comprising brxC/pglY, pglZ and at least one ofpglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, andbrxE, with the proviso that the BREX system does not comprise pglW.

The present invention further provides for a nucleic acid constructsystem comprising at least two nucleic acid constructs expressing a BREXsystem comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxDand brxHI.

Thus according to specific embodiments, the nucleic acid constructsystem comprises an individual nucleic acid construct for each BREXsystem pgl and/or brx gene.

According to other specific embodiments a single construct comprises anumber of BREX system pgl and/or brx genes.

Cis acting regulatory sequences include those that direct constitutiveexpression of a nucleotide sequence as well as those that directinducible expression of the nucleotide sequence only under certainconditions.

According to specific embodiments, the nucleic acid construct includes apromoter sequence for directing transcription of the polynucleotidesequence in the cell in a constitutive or inducible manner.

Constitutive promoters suitable for use with some embodiments of theinvention are promoter sequences which are active under mostenvironmental conditions and most types of cells such as thecytomegalovirus (CMV) and Rous sarcoma virus (RSV). Inducible promoterssuitable for use with some embodiments of the invention include forexample the tetracycline-inducible promoter (Zabala M. et al., CancerRes. 2004, 64(8): 2799-804) or pathogen-inducible promoters. Suchpromoters include those from pathogenesis-related proteins (PRproteins), which are induced following infection by a pathogen.

According to specific embodiments the promoter is a bacterial nucleicacid (e.g., expression) construct.

A bacterial promoter is any DNA sequence capable of binding bacterialRNA polymerase and initiating the downstream (3′) transcription of acoding sequence into mRNA. A promoter can have a transcriptioninitiation region, which is usually placed proximal to the 5′ end of thecoding sequence. This transcription initiation region typically includesan RNA polymerase binding site and a transcription initiation site. Abacterial promoter can also have a second domain called an operator,which can overlap an adjacent RNA polymerase binding site at which RNAsynthesis begins. The operator permits negative regulated (inducible)transcription, as a gene repressor protein can bind the operator andthereby inhibit transcription of a specific gene. Constitutiveexpression can occur in the absence of negative regulatory elements,such as the operator. In addition, positive regulation can be achievedby a gene activator protein binding sequence, which, if present isusually proximal (5′) to the RNA polymerase binding sequence.

An example of a gene activator protein is the catabolite activatorprotein (CAP), which helps initiate transcription of the lac operon inEscherichia coli (Raibaud et al. (1984) Annu. Rev. Genet. 18:173).Regulated expression can therefore be either positive or negative,thereby either enhancing or reducing transcription. Other examples ofpositive and negative regulatory elements are well known in the art.Various promoters that can be included in the protein expression systeminclude, but are not limited to, a T7/LacO hybrid promoter, a trppromoter, a T7 promoter, a lac promoter, and a bacteriophage lambdapromoter. Any suitable promoter can be used to carry out the presentinvention, including the native promoter or a heterologous promoter.Heterologous promoters can be constitutively active or inducible. Anon-limiting example of a heterologous promoter is given in U.S. Pat.No. 6,242,194 to Kullen and Klaenhammer.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) (Chang etal. (1987) Nature 198:1056), and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) (Goeddel et al. (1980) Nucleic Acids Res. 8:4057; Yelverton et al.(1981) Nucleic Acids Res. 9:731; U.S. Pat. No. 4,738,921; EPOPublication Nos. 36.776 and 121.775). The beta-lactamase (bla) promotersystem (Weissmann, (1981) “The Cloning of Interferon and OtherMistakes,” in Interferon 3 (ed. 1. Gresser); bacteriophage lambda PL(Shimatake et al. (1981) Nature 292:128); the arabinose-inducible araBpromoter (U.S. Pat. No. 5,028,530); and T5 (U.S. Pat. No. 4,689,406)promoter systems also provide useful promoter sequences. See also Balbas(2001) Mol. Biotech. 19:251-267, where E. coli expression systems arediscussed.

In addition, synthetic promoters that do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or phage promoter can be joined with theoperon sequences of another bacterial or phage promoter, creating asynthetic hybrid promoter (U.S. Pat. No. 4,551,433). For example, thetac (Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl.Acad. Sci. 80:21) and trc (Brosius et al. (1985) J. Biol. Chem.260:3539-3541) promoters are hybrid trp-lac promoters comprised of bothtrp promoter and lac operon sequences that are regulated by the lacrepressor. The tac promoter has the additional feature of being aninducible regulatory sequence. Thus, for example, expression of a codingsequence operably linked to the tac promoter can be induced in a cellculture by adding isopropyl-1-thio-.beta.-D-galactoside (IPTG).Furthermore, a bacterial promoter can include naturally occurringpromoters of non-bacterial origin that have the ability to bindbacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The phage T7 RNA polymerase/promoter system is anexample of a coupled promoter system (Studier et al. (1986) J. Mol.Biol. 189:113; Tabor et al. (1985) Proc. Natl. Acad. Sci. 82:1074). Inaddition, a hybrid promoter can also be comprised of a phage promoterand an E. coli operator region (EPO Publication No. 267,851).

The nucleic acid construct can additionally contain a nucleotidesequence encoding the repressor (or inducer) for that promoter. Forexample, an inducible vector of the present invention can regulatetranscription from the Lac operator (LacO) by expressing the nucleotidesequence encoding the LacI repressor protein. Other examples include theuse of the lexA gene to regulate expression of pRecA, and the use oftrpO to regulate ptrp. Alleles of such genes that increase the extent ofrepression (e.g., laclq) or that modify the manner of induction (e.g.,lambda C1857, rendering lambda pL thermo-inducible, or lambda CI+,rendering lambda pL chemo-inducible) can be employed.

Various construct schemes can be utilized to express few genes from asingle nucleic acid construct. For example, the genes can beco-transcribed as a polycistronic message from a single promotersequence of the nucleic acid construct. To enable co-translation of allthe genes from a single polycistronic message, the differentpolynucleotide segments can be transcriptionally fused via a linkersequence including an internal ribosome entry site (IRES) sequence whichenables the translation of the polynucleotide segment downstream of theIRES sequence. In this case, a transcribed polycistronic RNA moleculeincluding the coding sequences of all the genes will be translated fromboth the capped 5′ end and the internal IRES sequence of thepolycistronic RNA molecule to thereby produce the whole BREX system.

Alternatively, each two polynucleotide segments can be translationallyfused via a protease recognition site cleavable by a protease expressedby the cell to be transformed with the nucleic acid construct. In thiscase, a chimeric polypeptide translated will be cleaved by the cellexpressed protease to thereby generate the whole BREX system.

Still alternatively, the nucleic acid construct of some embodiments ofthe invention can include at least two promoter sequences each being forseparately expressing a specific pgl or brx. These at least twopromoters which can be identical or distinct can be constitutive, tissuespecific or regulatable (e.g. inducible) promoters functional in one ormore cell types.

The nucleic acid construct (also referred to herein as an “expressionvector” or a “vector”) of some embodiments of the invention includesadditional sequences which render this vector suitable for replicationand integration in prokaryotes, eukaryotes, or preferably both (e.g.,shuttle vectors). In addition, typical vectors may also contain atranscription and translation initiation sequence, transcription andtranslation terminator and a polyadenylation signal. By way of example,such constructs will typically include a 5′ LTR, a tRNA binding site, apackaging signal, an origin of second-strand DNA synthesis, and a 3′ LTRor a portion thereof.

When secretion of the polypeptides is desired the polynucleotides of theinvention can be expressed as fusion polypeptides comprising the nucleicacid sequence encoding the PGL or BRX gene ligated in frame to a nucleicacid sequence encoding a signal peptide that provides for secretion.

DNA encoding suitable signal sequences can be derived from genes forsecreted bacterial proteins, such as the E. coli outer membrane proteingene (ompA) (Masui et al. (1983) FEBS Lett. 151(1):159-164; Ghrayeb etal. (1984) EMBO J. 3:2437-2442) and the E. coli alkaline phosphatasesignal sequence (phoA) (Oka et al. (1985) Proc. Natl. Acad. Sci.82:7212). Other prokaryotic signals include, for example, the signalsequence from penicillinase, Ipp, or heat stable enterotoxin II leaders.

According to a specific embodiment, the nucleic acid construct comprisesa plurality of cloning sites for ligating a nucleic acid sequence of theinvention such that it is under transcriptional regulation of theregulatory regions.

Selectable marker genes that ensure maintenance of the vector in thecell can also be included in the expression vector. Preferred selectablemarkers include those which confer resistance to drugs such asampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), andtetracycline (Davies et al. (1978) Annu. Rev. Microbiol. 32:469).Selectable markers can also allow a cell to grow on minimal medium, orin the presence of toxic metabolite and can include biosynthetic genes,such as those in the histidine, tryptophan, and leucine biosyntheticpathways.

In the construction of the expression vector, the promoter is preferablypositioned approximately the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

Other than containing the necessary elements for the transcription andtranslation of the inserted coding sequence, the expression construct ofsome embodiments of the invention can also include sequences engineeredto enhance stability, production, purification, yield or toxicity of theexpressed polypeptide.

Where appropriate, the polynucleotides may be optimized for increasedexpression in the transformed organism. For example, the polynucleotidescan be synthesized using preferred codons for improved expression.

Various methods known within the art can be used to introduce theexpression vector of some embodiments of the invention into cells. Suchmethods are generally described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Springs Harbor Laboratory, New York (1989,1992), in Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic GeneTherapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., GeneTargeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey ofMolecular Cloning Vectors and Their Uses, Butterworths, Boston Mass.(1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] andinclude, for example, stable or transient transfection, natural orinduced transformation, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

Exemplary methods of introducing expression vectors into bacterial cellsinclude for example conventional transformation or transfectiontechniques, or by phage-mediated infection. As used herein, the terms“transformation”, “transduction”, “conjugation”, and “protoplast fusion”are intended to refer to a variety of art-recognized techniques forintroducing foreign nucleic acid (e.g., DNA) into a cell, such ascalcium chloride co-precipitation.

Introduction of nucleic acids by phage infection offers severaladvantages over other methods such as transformation, since highertransfection efficiency can be obtained due to the infectious nature ofphages. These methods are especially useful for rendering bacteria moresensitive to phage attack for antibiotics purposes as further describedhereinbelow.

It will be appreciated the BREX polypeptides can be introduced directlyinto the cell (e.g., bacterial cell) and not via recombinant expressionto confer resistance. The term “polypeptide” as used herein encompassesnative peptides (either degradation products, synthetically synthesizedpeptides or recombinant peptides) and peptidomimetics (typically,synthetically synthesized peptides), as well as peptoids andsemipeptoids which are peptide analogs, which may have, for example,modifications rendering the peptides more stable while in a body or morecapable of penetrating into cells. Such modifications include, but arenot limited to N terminus modification, C terminus modification, peptidebond modification, backbone modifications, and residue modification.Methods for preparing peptidomimetic compounds are well known in the artand are specified, for example, in Quantitative Drug Design, C. A.Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which isincorporated by reference as if fully set forth herein.

The polypeptides of the present invention may be synthesized by anytechniques known to those skilled in the art of peptide synthesis, forexample but not limited to recombinant DNA techniques or solid phasepeptide synthesis.

Thus, regardless of the method of introduction, the present teachingsprovide for an isolated cell (e.g., bacterial cell) which comprises aheterologous BREX system, as described herein.

According to specific embodiments, the isolated cell is transformed ortransfected with the above-mentioned nucleic acid construct or nucleicacid construct system.

According to an aspect of the present invention, there is provided anisolated cell genetically modified to express a BREX system selectedfrom the group consisting of

(1) brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHI,brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREXsystem does not comprise pglW,

(2) brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI,

(3i) brxA, brxB, brxC/pglY, pglX, pglZ and brxL.

(3ii) brxA, brxB, (brxC/pglY)_(x2), pglX, pglZ and brxHII.

(3iii) brxE, brxA, brxB, brxC/pglY, pglX, pglZ, brxD and brxHI,

(3iv) brxF, brxC/pglY, pglXI, brxHII, pglZ and brxA,

(3v) pglW, pglX, brxC/pglY, pglZ, brxD and brxHI, or

(3vi) brxP, brxC/pglY, pglZ and brxL.

According to another aspect of the present invention there is providedan isolated cell genetically modified to express a BREX systempolypeptide selected from the group consisting of pglX, brxP, brxHI,brxHII, brxL, brxD, brxA, brxB, brxF, and brxE.

According to specific embodiments there is provided an isolated cellgenetically modified to express a BREX system polypeptide comprising anamino acid sequence of a COG0515 domain, said polypeptide conferringresistance to a first cycle phage infection.

According to specific embodiments there is provided an isolated cellgenetically modified to express a BREX system polypeptide comprising anamino acid sequence of a pfam13659 domain, said polypeptide conferringresistance to a first cycle phage infection.

According to specific embodiments there is provided an isolated cellgenetically modified to express a BREX system polypeptide comprising anamino acid sequence of DUF2791 and DUF499 domains, said polypeptideconferring resistance to a first cycle phage infection.

According to specific embodiments there is provided an isolated cellgenetically modified to express a BREX system polypeptide comprising anamino acid sequence of a pfam08665 domain, said polypeptide conferringresistance to a first cycle phage infection.

According to specific embodiments there is provided an isolated cellgenetically modified to express a pglW polypeptide with the proviso thatsaid pglW polypeptide is not SEQ ID NO: 6110.

According to specific embodiments there is provided an isolated cellgenetically modified to express a pglX polypeptide with the proviso thatsaid pglX polypeptide is not SEQ ID NO: 2949.

According to specific embodiments there is provided an isolated cellgenetically modified to express a brxC/pglY polypeptide with the provisothat said brxC/pglY polypeptide is not SEQ ID NO: 802.

According to specific embodiments there is provided an isolated cellgenetically modified to express a pglZ polypeptide with the proviso thatsaid pglZ polypeptide is not SEQ ID NO: 1890.

According to specific embodiment the isolated cell (e.g., bacterialcell) does not express a BREX system endogenously.

The term “endogenous” as used herein, refers to the expression of thenative gene in its natural location and expression level in the genomeof an organism.

The expression of the polynucleotide can be episomal or integrated intothe chromosome of the cell.

According to specific embodiments the isolated cell is resistant to afirst cycle phage infection.

According to specific embodiments the isolated cell is resistant tolytic phage.

According to specific embodiments the isolated cell is resistant totemperate (also referred as lysogenic) phage.

According to a specific embodiment the isolated cell is resistant tophage lysogeny.

According to another specific embodiment the isolated cell is resistantto phage DNA replication.

According to specific embodiments the isolated cell is a microbial cellsuch as a bacterial cell.

As used herein, the term “bacteria” refers to all prokaryotes andincludes both bacteria and archaea.

Indeed, it is intended that any bacterial species (e.g., which does notexpress a PYZA system) will find use in the present invention. Thus, thebacteria may be for example gram-positive or gram-negative bacteria.

The phrase “Gram-positive bacteria” as used herein refers to bacteriacharacterized by having as part of their cell wall structurepeptidoglycan as well as polysaccharides and/or teichoic acids and arecharacterized by their blue-violet color reaction in the Gram-stainingprocedure. Representative Gram-positive bacteria include: Actinomycesspp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum,Clostridium perfringens. Clostridium spp., Clostridium tetani,Corynebacterium diphtheriae. Corvnebacteriwnum jeikeium, Enterococcusfaecalis, Enterococcus faecium, Ervsipelothrix rhusiopathiae,Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum,Leuconostoc spp., Mycobacterium abcessus, Mycobacterium avium complex.Mycobacterium chelonae, Mycobacterium fortuitum. Mycobacteriumhaemophilium, Mycobacterium kansasii, Mycobacterium leprae,Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacteriumsmegmatis. Mycobacterium terrae, Mycobacterium tuberculosis,Mycobacterium ulcerans, Nocardia spp., Peptococcus niger,Peptostreptococcus spp., Proprionibacterium spp., Staphylococcus aureus,Staphylococcus auricularis, Staphylococcus capitis, Staphylococcuscolmii, Staphylococcus epidermidis, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus lugdanensis, Staphylococcussaccharolyticus, Staphylococcus saprophyticus, Staphylococcusschleiferi, Staphylococcus similans, Staphylococcus warneri,Staphylococcus xylosus. Streptococcus agalactiae (group Bstreptococcus), Streptococcus anginosus, Streptococcus bovis,Streptococcus canis. Streptococcus equi, Streptococcus milleri,Streptococcus mitior, Streptococcus mutans. Streptococcus pneumoniae,Streptococcus pyogenes (group A streptococcus), Streptococcussalivarius, Streptococcus sanguis.

The term “Gram-negative bacteria” as used herein refers to bacteriacharacterized by the presence of a double membrane surrounding eachbacterial cell. Representative Gram-negative bacteria includeAcinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans,Aeromonas hydrophila, Alcaligenes xvlosoxidans, Bacteroides. Bacteroidesfragilis, Bartonella bacilliformis, Bordetella spp., Borreliaburgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp.,Chalmydia pneumoniae, Chlamndia psittaci. Chlamydia trachomatis,Chromobacterium violaceum, Citrobacter spp., Eikenella corrodens,Enterobacter aerogenes, Escherichia coli, Flavobacteriummeningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilusspp., Helicobacter pylori, Klebsiella spp., Legionella spp., Leptospiraspp., Moraxella catarrhalis, Morganella morganii, Mycoplasma pneumoniae,Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida,Plesiomonas shigelloides. Prevotella spp., Proteus spp., Providenciarettgeri, Pseudomonas aeruginosa, Pseudomonas spp., Rickettsiaprowazekii, Rickettsia rickettsii, Rochalimaea spp., Salmonella spp.,Salmonella typhi, Serratia marcescens, Shigella spp., Treponemacarateum, Treponema pallidum, Treponema pallidum endemicum, Treponemapertenue, Veillonella spp., Vibrio cholerae, Vibrio vulnificus, Yersiniaenterocolitica, Yersinia pestis.

According to specific embodiments the bacteria is a species selectedfrom the group consisting of Escherichia, Shigella, Salmonella, Erwinia,Yersinia, Bacillus, Vibrio, Legionella, Pseudomonas, Neisseria,Bordetella, Helicobacter, Listeria, Agrobacterium, Staphylococcus,Streptococcus, Enterococcus, Clostridium, Corynebacterium,Mycobacterium, Treponema, Borrelia, Francisella, Brucella,Campylobacter, Klebsiella, Frankia, Bartonella, Rickettsia, Shewanella,Serratia, Enterobacter, Proteus, Providencia, Brochothrix,Bifidobacterium, Brevibacterium, Propionibacterium. Lactococcus,Lactobacillus, Pediococcus, Leuconostoc. Oenococcus, andPropionibacterium species.

Additionally, or alternatively the bacteria may be useful in themanufacture of dairy and fermentation processing such as, but notlimited to, milk-derived products, such as cheeses, yogurt, fermentedmilk products, sour milks, and buttermilk.

According to specific embodiments the bacteria is a lactic bacteria. Asused herein the term “lactic acid bacteria” refers to Gram positive,microaerophillic or anaerobic bacteria which ferment sugar with theproduction of acids including lactic acid as the predominantly producedacid, acetic acid, formic acid and propionic acid.

According to specific embodiments the bacteria is a species selectedfrom the group of the industrially most useful lactic acid bacteriaconsisting of Lactococcus species, Streptococcus species, Lactobacillusspecies, Leuconostoc species, Oenococcus species, Pediococcus speciesand Bifidobacterium species and Propionibacterium species.

As used herein, the term “phage” or “bacteriophage” refers to a virusthat selectively infects one or more bacterial species. Many phages arespecific to a particular genus or species or strain of bacteria.

According to specific embodiments, the phage is virulent to thebacteria.

According to some embodiments, the phage is a lytic phage.

According to other embodiments, the phage is temperate (also referred toas lysogenic).

A lytic phage is one that follows the lytic pathway through completionof the lytic cycle, rather than entering the lysogenic pathway. A lyticphage undergoes viral replication leading to lysis of the cell membrane,destruction of the cell, and release of progeny phage particles capableof infecting other cells.

A temperate phage is one capable of entering the lysogenic pathway, inwhich the phage becomes a dormant, passive part of the cell's genomethrough prior to completion of its lytic cycle.

Exemplary phages which fall under the scope of the invention include,but are not limited to, phages that belong to any of the following virusfamilies: Corticoviridae, Cystoviridae, Inoviridae, Leviviridae,Microviridae, Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae.

According to specific embodiments the phage is selected from the groupconsisting of SPβ, SP16, Zeta, Φ3T, and SPO2.

According to other specific embodiments the phage is not Φ105, rho10 andrho14.

According to specific embodiments, the lytic phage is SPO1 and/or SP82G.

According to specific embodiments, phage that infect bacteria that arepathogenic to plants and/or animals (including humans) find particularuse.

According to specific embodiments, the resistance of a cell against aphage is improved as compared to a cell of the same species which wasnot treated according to the present teachings (i.e., with a BREXsystem).

The lysogenic activity of a phage can be assessed in multiple ways,including but not limited to PCR and DNA sequencing.

The DNA replication activity of a phage can be assessed in multipleways, including but not limited to DNA sequencing and southern blotanalysis.

The lytic activity of a phage can be assessed in multiple ways,including but not limited to optical density, plaque assay, and livingdye indicators.

The lytic activity of a phage can be measured indirectly by followingthe decrease in optical density of the bacterial cultures owing tolysis. This method involves introduction of phage into a fluid bacterialculture medium. After a period of incubation, the phage lyses thebacteria in the broth culture resulting in a clearing of the fluidmedium resulting in decrease in optical density.

Another method, known as the plaque assay, introduces phage into a fewmilliliters of soft agar along with some bacterial host cells. This softagar mixture is laid over a hard agar base (seeded-agar overlay). Thephage adsorb onto the host bacterial cells, infect and lyse the cells,and then begin the process anew with other bacterial cells in thevicinity. After 6-24 hours, zones of clearing on the plate known asplaques, are observable within the lawn of bacterial growth on theplate. Each plaque represents a single phage particle in the originalsample.

Yet another method is the one-step phage growth curve which allowsdetermining the production of progeny virions by cells as a function oftime after infection. The assay is based on the fact that cells in theculture are infected simultaneously with a low number of phages so thatno cell can be infected with more than one phage. At various timeintervals, samples are removed for a plaque assay allowing quantitativedetermination of the number of phages present in the medium.

Other methods use for example redox chemistry, employing cellrespiration as a universal reporter. During active growth of bacteria,cellular respiration reduces a dye (e.g., tetrazolium dye) and producesa color change that can be measured in an automated fashion. On theother hand, successful phage infection and subsequent growth of thephage in its host bacterium results in reduced bacterial growth andrespiration and a concomitant reduction in color.

Thus, the polynucleotides, polypeptides and nucleic acid constructs ofthe present invention can be used in conferring phage resistance.

As used herein, “confers phage resistance” refers to an increase of atleast 10% in bacterial resistance towards a phage, as may be manifestedin viability. According to a specific embodiment, the increase is in atleast 20%, 30%, 40% or even higher say, 50%, 60%, 70%, 80%, 90% or morethan 100%.

For the same culture conditions the bacterial susceptibility towards aphage of the present invention is generally expressed in comparison tothe wild-type bacteria. As used herein, the phrase “increased resistancetowards a phage” means that the level of phage infection and/ormultiplication in the bacteria does not cause a deleterious effect tothe bacteria e.g., growth arrest or death.

In some embodiments, the bacteria have about 100-100.000 times lowerefficiency of plaquing ([EOP]=10-2), about 1000 times lower EOP(EOP=10⁼³), 10,000 times lower EOP (EOP=10-4), or 100,000 times lowerEOP (EOP=10-5). In some embodiments, the level of phage multiplicationin a culture is measured after about 6-14 hours incubation of theculture, e.g., after about 12 hours, after about 9 hours, after about 8hours after about 7 hours, or after about 6 hours.

Thus, according to specific embodiments there is provided a method ofprotecting bacteria from phage attack, the method comprising introducinginto or expressing in the bacteria a BREX system, thereby protecting thebacteria from phage attack.

According to specific embodiments the bacteria does not express a BREXsystem endogenously.

Various modalities may be used to introduce or express the BREX systemin the bacteria.

Thus, according to specific embodiments, the method is effected byexpressing in the bacteria, the isolated polynucleotides, nucleic acidconstruct or construct system or alternatively introducing the BREXpolypeptides as described herein to confer protection.

According to another embodiment the BREX system is introduced into thebacteria via a transmissible genetic element in a process of bacterialconjugation.

As used herein, the phrase “bacterial conjugation” refers to a directtransfer of genetic material between bacterial cells by cell-to-cellcontact or by bridge-like connection between the cells. Duringconjugation the donor bacteria provides a transmissible genetic element,typically a plasmid or a transposon. The transfer of the transmissiblegenetic element tale advantage of the complementary nature of doublestranded DNA. Thus, one strand of the transmissible genetic element istransferred and the other remains in the original bacteria. Both strandshave the complementary stranded added so that each bacteria ends up witha complete transmissible element.

According to a specific embodiment, there is provided a method ofprotecting first bacteria from phage attack, the method comprisingcontacting the first bacteria with second bacteria which expresses on atransmissible genetic element a BREX system, wherein the first bacteriaand the second bacteria are non identical; thereby protecting thebacteria from phage attack.

As used herein, the term “contacting” refers to the step of incubationof the bacterial cell (e.g., first bacteria) with a substance or cell(e.g., second bacteria) such that the substance or a substance containedin the cell affects phage resistance of the bacterial cell.

According to specific embodiments the first bacteria does not express aBREX system endogenously.

As used herein the phrase “transmissible genetic element” refers to anucleic acid sequence that can be transferred naturally from onebacteria to another.

According to specific embodiments the transmissible genetic elementcomprises a conjugative genetic element or a conjugative plasmid ormobilizable genetic element.

As used herein, a “conjugative plasmid” refers to a plasmid that istransferred from one bacterial cell to another during conjugation.

As used herein, the term “mobilizable element” refers to a transposon,which is a DNA sequence that can change its position within the genome.

According to a specific embodiment, the first bacteria is theindustrially valuable bacteria such as those used for fermentation asdescribed above.

Thus, following the above teachings there is provided an isolatedbacteria comprising a nucleic acid sequence encoding a BREX system and atransmissible genetic element expressing the BREX system, wherein theisolated bacteria does not endogenously express the BREX system andwherein the BREX system comprises brxC/pglY, pglZ and at least one ofpglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, andbrxE, with the proviso that the BREX system does not comprise pglW, orcomprises brxC/pglY, pglZ, pglX, pglW and at least one of brxD andbrxHI.

Cultures, and starter cultures, in particular are used extensively inthe food industry in the manufacture of fermented products includingmilk products (e.g., yogurt, buttermilk, and cheese), meat products,bakery products, wine, and vegetable products. The preparation ofcultures is labor intensive, occupying much space and equipment, andthere is a considerable risk of contamination with spoilage bacteriaand/or phages during the propagation steps. The failure of bacterialcultures due to phage infection and multiplication is a major problemwith the industrial use of bacterial cultures. There are many differenttypes of phages and new strains continue to emerge. Indeed, despiteadvances in culture development, there is a continuing need to improvecultures for use in industry.

Thus, according to an aspect of the present invention, there is provideda method for preparing a food, food additive, feed, nutritionalsupplement, probiotic supplement, a personal care product, a health careproduct, and a veterinary product comprising adding to the food, foodadditive, feed, nutritional supplement, probiotic supplement, a personalcare product, a health care product, and a veterinary product theisolated BREX system polynucleotide, the BREX system construct, theisolated cell or the isolated bacteria of the present invention, therebypreparing the food, food additive, feed, nutritional supplement,probiotic supplement, personal care product, health care product, andveterinary product.

Thus, following the above teachings there is provided a food, foodadditive, feed, nutritional supplement, probiotic supplement, a personalcare product, a health care product, and a veterinary product comprisingthe isolated BREX system polynucleotide, the BREX system construct, theisolated cell or the isolated bacteria of the present invention.

According to another aspect of the present invention, there is provideda method for preparing a food, food additive, feed, nutritionalsupplement, probiotic supplement, a personal care product, a health careproduct, and a veterinary product comprising adding to the food, foodadditive, feed, nutritional supplement, probiotic supplement, a personalcare product, a health care product, and a veterinary product a bacteriawhich expresses on a transmissible genetic element a BREX systemcomprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI,brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso thatthe BREX system does not comprise pglW, or comprising brxC/pglY, pglZ,pglX, pglW and at least one of brxD and brxHI, thereby preparing thefood, food additive, feed, nutritional supplement, probiotic supplement,personal care product, health care product, and veterinary product.

According to specific embodiments the food or feed is a dairy product.

The preparation of starter cultures of such bacteria, and methods offermenting substrates, particularly food substrates such as milk, can becarried out in accordance with known techniques, including but notlimited to those described in Mayra-Makinen and Bigret (1993) LacticAcid Bacteria; Salminen and vonWright eds. Marcel Dekker, Inc. New York.65-96; Sandine (1996) Dairy Starter Cultures Cogan and Accolas eds. VCHPublishers, New York. 191-206; Gilliland (1985) Bacterial StarterCultures for Food. CRC Press, Boca Raton. Fla.

The term “fermenting” refers to the energy-yielding, metabolic breakdownof organic compounds by microorganisms that generally proceeds underanaerobic conditions and with the evolution of gas.

Products produced by fermentation which have been known to experiencephage infection, and the corresponding infected fermentation bacteria,include cheddar and cottage cheese (Lactococcus lactis subsp. lactis,Lactococcus lactis subsp. cremoris), yogurt (Lactobacillus delbrueckiisubsp. bulgaricus, Streptococcus thermophilus). Swiss cheese (S.thermophilus, Lactobacillus lactis, Lactobacillus helveticus), bluecheese (Leuconostoc cremoris), Italian cheese (L. bulgaricus, S.thermophilus), viili (Lactococcus lactis subsp. cremoris. Lactococcuslactis subsp. lactis biovar diacetylactis, Leuconostoc cremoris), yakult(Lactobacillus casei), casein (Lactococcus lactis subsp. cremoris),natto (Bacillus subtilis var. natto), wine (Leuconostoc oenos), sake(Leuconostoc mesenteroides), polymyxin (Bacillus polymyxa), colistin(Bacillus colistrium), bacitracin (Bacillus licheniformis), L-glutamicacid (Brevibacterium lactofermentum, Microbacterium ammoniaphilum), andacetone and butanol (Clostridium acetobutylicum. Clostridiumsaccharoperbutvlacetonicum).

The present inventors have uncovered that transformation of a Bacillussubtilis strain with a non-complete type 1 BREX (i.e. not expressingpglX) does not confer phage resistance. In addition it was alsodiscovered that a frame shift mutation in a BREX gene (i.e., pglX) inone of the Bacillus subtilis strains transformed with type 1 BREXresulted in aberrant BREX system that was not active against any of thetested phages, indicating that down regulation of a BREX gene can rendera bacteria resistant to phage infection. These results suggest the useof anti BREX agents as a method to induce phage sensitivity.

As used herein, “inducing phage sensitivity” refers to an increase of atleast 10% in bacterial susceptibility towards a phage, as may bemanifested in growth arrest or death. According to a specificembodiment, the increase is in at least 20%, 30%, 40% or even highersay, 50%, 60%, 70%, 80%, 90% or more than 100%.

For the same culture conditions, the bacterial susceptibility towards aphage of the present invention is generally expressed in comparison tothe wild-type bacteria. As used herein, the phrase “increasedsusceptibility towards a phage” means that the level of phage infectionand/or multiplication in the bacteria cause a deleterious effect to thebacteria e.g., growth arrest or death.

Thus, according to further aspect of the present invention, there isprovided a method of inducing phage sensitivity in a bacterial cell, themethod comprising contacting a bacterial cell which expresses a BREXsystem comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP,brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the provisothat the BREX system does not comprise pglW, or comprising brxC/pglY,pglZ, pglX, pglW and at least one of brxD and brxHI; with an anti BREXsystem agent capable of down regulating a BREX gene selected from thegroup consisting of brxC/pglY, pglZ, pglX, pglX, brxP, brxHI, brxHI,brxL, brxD, brxA, brxB, brxF, brxE, and pglW, thereby inducingsensitivity of the bacterial cell to phage infection.

As used herein the phrase “anti BREX system agent” is an agent capableof specifically inhibiting or “silencing” the expression of a targetBREX gene or alternatively specifically impairs the functionality of thetarget BREX protein. According to specific embodiments the anti BREXsystem agent is directed against pglX. For example, the anti-BREX systemmay interfere with pglX expression (as described hereinbelow) or in itsDNA methyltransferase function by the use of common inhibitors of suchan enzyme e.g., 5-Azacytidine. Decitabine Zebularine, RG108, Hydralazinehydrochloride, and Psammaplin A.

According to other specific embodiments the anti BREX system agent isdirected against brxC/pglY or pglZ.

Down regulation of BREX system can be effected on the genomic and/or thetranscript level using a variety of molecules which interfere withtranscription and/or translation (e.g., RNA silencing agents), or on theprotein level using e.g., aptamers, small molecules and inhibitorypeptides, antagonists, enzymes that cleave the polypeptide and the like.

According to specific embodiments the anti BREX system agent is selectedfrom the group consisting of a nucleic acid suitable for silencingexpression, aptamers, small molecules and inhibitory peptides.

As used herein the phrase “nucleic acid suitable for silencingexpression” refers to regulatory mechanisms mediated by nucleic acidmolecules which result in the inhibition or “silencing” of theexpression of a corresponding protein-coding gene. Numerous methods areknown in the art for gene silencing in prokaryotes. Examples include butare not limited to U.S. Patent Application 20040053289 which teaches theuse of si hybrids to down-regulate prokaryotic genes, and

U.S. Patent Application PCT/US09/69258 which teaches the use of CRISPRto downregulate prokaryotic genes. Alternatively the inhibition can becarried out at the protein level which interferes with protein activity,such as by the use of aptamers. Various methods are known in the artwhich can be used to design protein specific aptamers. The skilledartisan can employ SELEX (Systematic Evolution of Ligands by ExponentialEnrichment) for efficient selection as described in Stoltenburg R,Reinemann C, and Strehlitz B (Biomolecular engineering (2007)24(4):381-403).

As used herein an “aptamer” refers to double stranded DNA or singlestranded RNA molecule that binds to specific molecular target, such as aprotein.

Alternatively or additionally, small molecule or peptides can be usedwhich interfere with the BREX protein function (e.g., catalytic orinteraction).

Specifically, contacting is effected such that the positioning of theanti BREX system agent is in direct or indirect contact with thebacterial cell. Thus, the present invention contemplates both applyingthe anti BREX system agents of the present invention to a desirablesurface and/or directly to the bacterial cells.

According to another embodiment the surface is comprised in a biologicaltissue, such as for example, mammalian tissues e.g. the skin.

It will be appreciated that the bacteria may be comprised inside aparticular organism, (e.g. intracellularly or extracellularly) forexample inside a mammalian body or inside a plant. In this case, thecontacting may be effected by administering the anti BREX agents per seor by transfecting the cells of the organism with the anti BREX agentsof the present invention.

Thus, according to a specific embodiment contacting with an anti BREXsystem agent is effected in-vivo.

According to another specific embodiment contacting with an anti BREXsystem agent is effected ex-vivo.

According to another specific embodiment contacting with an anti BREXsystem agent is effected in-vitro.

According to specific embodiments, there is provided an isolatedbacteria generated by contacting bacteria with anti BREX system agentin-vitro or ex-vivo.

According to some embodiments, a BREX system or an anti-BREX systemagent is provided in a formulation suitable for cell penetration thatenhances intracellular delivery of BREX system.

Any suitable penetrating agent for enhancing penetration of BREX systemor anti BREX system agent to cell (e.g., bacteria) may be used, as knownby those of skill in the art. Examples include but are not limited to:

Phages—Phages offer several advantages including lateral infection,higher efficiency of transformation, and targeting to, and propagationin, specific bacteria.

Cell-Penetrating Peptides (CPPs)—CPPs, for example TAT (transcriptionactivator from HIV-1) are short peptides (≤40 amino acids), with theability to gain access to the interior of almost any cell. They arehighly cationic and usually rich in arginine and lysine amino acids.They have the exceptional property of carrying into the cells a widevariety of covalently and noncovalently conjugated cargoes such asproteins, oligonucleotides, and even 200 nm liposomes. Protocols forproducing CPPs-cargos conjugates and for infecting cells with suchconjugates can be found, for example L. Theodore et al. [The Journal ofNeuroscience, (1995) 15(11): 7158-7167], Fawell S, et al. [Proc NatlAcad Sci USA, (1994) 91:664-668], and Jing Bian et al. [CirculationResearch. (2007) 100: 1626-1633].

The expression level and/or activity level of the BREX system expressedin the cells of some embodiments of the invention can be determinedusing methods known in the arts, e.g. but not limited to selectablemarker gene, Northern blot analysis, PCR analysis, DNA sequencing, RNAsequencing, Western blot analysis, and Immunohistochemistry.

According to another aspect of the present invention, there is provideda method of treating a microbial infection in a subject in need thereof,the method comprising contacting the bacteria with an anti BREX systemagent capable of down regulating a BREX gene selected from the groupconsisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI, brxHII, brxL,brxD, brxA, brxB, brxF, brxE, and pglW, thereby treating the infection.

As used herein, the term “treating” refers to curing, reversing,attenuating, alleviating, minimizing, suppressing or halting thedeleterious effects of a pathogen infection.

As used herein, the phrase “subject in need thereof” includes mammals,preferably human beings at any age which suffer from pathogen infection.

The anti BREX system agent may be used alone or together with additionalantimicrobial agents (e.g. phage therapy, antibiotic and/or additionalanti microbial peptides).

According to specific embodiments the methods of the present inventionfurther comprise administering to the subject a phage therapy.

According to other specific embodiments the methods of the presentinvention further comprise administering to the subject an antibiotic.

Exemplary antibiotics include, but are not limited to aminoglycosideantibiotics, cephalosporins, quinolone antibiotics, macrolideantibiotics, penicillins, sulfonamides, tetracyclines and carbapenems.It will be appreciated that since the polypeptides of embodiments ofthis invention enhance the antibacterial effect of the antibiotic, dosesof the antibiotic may be lower (e.g. 20% lower, 30% lower, 40% lower,50% lower, 60% lower, 70% lower, 80% lower or even 90% lower than thosecurrently in use.

The BREX system or the anti-BREX system agent of some embodiments of theinvention can be administered to a starter culture, a fermentation vator an organism per se, or in a composition where it is mixed withsuitable carriers or excipients.

According to an aspect of the present invention there is provided aphage defense composition, comprising as an active ingredient a BREXsystem comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP,brxHII, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the provisothat the BREX system does not comprise pglW, or comprising brxC/pglY,pglZ, pglX, pglW and at least one of brxD and brxHI; and an acceptablecarrier or diluent.

According to another aspect of the present invention there is providedan anti-microbial composition comprising as active ingredient an antiBREX system agent capable of down regulating a BREX gene selected fromthe group consisting of brxC/pglY, pglZ, pglX, pglXI, brxP, brxHI,brxHII, brxL, brxD, brxA, brxB, brxF, brxE, and pglW, and an acceptablecarrier or diluent.

As used herein, the phrase “anti-microbial activity”, refers to anability to suppress, control, inhibit or kill a bacteria. Thus, forexample the anti-microbial activity may comprise bactericidal orbacteriostatic activity, or both.

According to specific embodiments the composition is a pharmaceuticalcomposition and the carrier is a pharmaceutically acceptable carrier.

The phrase “pharmaceutical composition” as used herein refers to apreparation of one or more of the active ingredients described hereinwith other chemical components such as physiologically suitable carriersand excipients. The purpose of a pharmaceutical composition is tofacilitate administration of a compound to an organism.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein, the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils, and polyethyleneglycols.

As used herein the term “active ingredient” refers to any one of BREXsystem polypeptide or polynucleotide, anti-BREX system agent capable ofdown regulating a BREX gene or cells generated according to the presentteachings, accountable for the biological effect.

Techniques for formulation and administration of drugs may be found inthe latest edition of “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., which is herein fully incorporated byreference and are further described herein below.

It will be appreciated that the polypeptides, polynucleotides, or otheragents of the present invention can be provided to the individual withadditional active agents to achieve an improved therapeutic effect ascompared to treatment with each agent by itself.

Exemplary additional agents include phage therapy, and antibiotics (e.g.rifampicin, chloramphenicol and spectinomycin).

According to specific embodiment the anti-microbial composition furthercomprises a phage.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, intraperitoneal, intranasal, orintraocular injections.

Conventional approaches for drug delivery to the central nervous system(CNS) include: neurosurgical strategies (e.g., intracerebral injectionor intracerebroventricular infusion); molecular manipulation of theagent (e.g., production of a chimeric fusion protein that comprises atransport peptide that has an affinity for an endothelial cell surfacemolecule in combination with an agent that is itself incapable ofcrossing the BBB) in an attempt to exploit one of the endogenoustransport pathways of the BBB; pharmacological strategies designed toincrease the lipid solubility of an agent (e.g., conjugation ofwater-soluble agents to lipid or cholesterol carriers); and thetransitory disruption of the integrity of the BBB by hyperosmoticdisruption (resulting from the infusion of a mannitol solution into thecarotid artery or the use of a biologically active agent such as anangiotensin peptide). However, each of these strategies has limitations,such as the inherent risks associated with an invasive surgicalprocedure, a size limitation imposed by a limitation inherent in theendogenous transport systems, potentially undesirable biological sideeffects associated with the systemic administration of a chimericmolecule comprised of a carrier motif that could be active outside ofthe CNS, and the possible risk of brain damage within regions of thebrain where the BBB is disrupted, which renders it a suboptimal deliverymethod.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, for oralingestion by a patient. Pharmacological preparations for oral use can bemade using a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The preparation of the present invention may also be formulated inrectal compositions such as suppositories or retention enemas, using,e.g., conventional suppository bases such as cocoa butter or otherglycerides.

The preparation of the present invention may also be formulated as atopical composition, such as a spray, a cream, a mouthwash, a wipe, afoam, a soap, an oil, a solution, a lotion, an ointment, a paste, a geland a patch.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of disease (e.g., bacterial infection) or prolong the survivalof the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro assays. For example, a dose can be formulated in animal modelsand such information can be used to more accurately determine usefuldoses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. [See e.g., Fingl, et al., (1975) “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1].

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of some embodiments of the invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions comprising a preparation of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition, as is further detailed above.

According to another aspect there is provided an article of manufactureor a kit identified for killing a bacteria comprising a packagingmaterial packaging an anti BREX system agent capable of down regulatinga BREX gene selected from the group consisting of brxC/pglY, pglZ, pglX,pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, brxE, andpglW, and a phage.

According to specific embodiments the anti BREX system agent and thephage are packaged in separate containers.

According to yet other specific embodiments the anti BREX system agentand the phage are in c-formulation.

According to further aspect of the present invention there is provided amethod of screening for identifying phage useful for infecting abacteria, the method comprising:

(a) contacting a phage with a bacteria expressing BREX system comprisingbrxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII,brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that the BREXsystem does not comprise pglW or comprising brxC/pglY, pglZ, pglX, pglWand at least one of brxD and brxHI;

(b) monitoring phage sensitivity of the bacteria, wherein an increase inphage sensitivity of the bacteria in the presence of the phage comparedto phage sensitivity in the absence of the phage is indicative of aphage useful for infecting the bacteria.

The method comprising further isolating the phage characterizing it interms of sequencing and compatibility with phages species and theability to infect different bacterial species.

Tables 2-8 and 10-16 below demonstrate the six types of BREX system in adiverse array of bacteria and archaea genomes.

TABLE 2 BREX type 1 systems Genomic Genomic Start End Methylase RepliconOrganism Point Point BrxA* BrxB* BrxC* PglX* PglZ* BrxL* state TypeAcidiphilium 497519 512043 YP_004282676.1/ YP_004282675.1/YP_004282674.1/ YP_004282672.1/ YP_004282670.1/ YP_004282669.1/ Splitchromosome multivorum AIU301 4953 5570 617 2766 1716 4300 methylase.uid63345 YP_004282673.1/ 2767 Acidithiobacillus 2361379 2377748YP_004784791.1/ YP_004784792.1/ YP_004784793.1/ YP_004784794.1/YP_004784799.1/ YP_004784800.1/ Split chromosome ferrivorans SS3uid67387 4954 5571 618 2768 1717 4301 methylase. YP_004784796.1/ 2769Acinetobacter baumannii 53842 68427 YP_001708756.1/ YP_001708755.1/YP_001708754.1/ YP_001708753.1/ YP_001708751.1/ YP_001708750.1/ plasmidAYE uid61637 4955 5572 619 2770 1718 4302 Anaeromyxobacter 36883963701032 YP_466441.1/ YP_466440.1/ YP_466439.1/ YP_466438.1/ YP_466437.1/YP_466436.1/ chromosome dehalogenans 2CP C 4957 5574 621 2772 1720 4303uid58135 Aromatoleum 3091305 3115716 YP_159986.1/ YP_159987.1/YP_159988.1/ YP_159991.1/ YP_160004.1/ YP_160005.1/ chromosomearomaticum EbN1 4958 5575 622 2773 1721 4304 uid58231 BurkholderiaCCGE1001 3119206 3136704 YP_004229224.1/ YP_004229223.1/ YP_004229222.1/YP_004229221.1/ YP_004229217.1/ YP_004229216.1/ chromosome uid42975 49635580 627 2777 1726 4309 Burkholderia gladioli 1574842 1588056YP_004360051.1/ YP_004360052.1/ YP_004360053.1/ YP_004360054.1/YP_004360055.1/ YP_004360056.1/ chromosome BSR3 uid66301 4964 5581 6282778 1727 4310 Burkholderia 2201162 2216554 YP_001119856.1/YP_001119855.1/ YP_001119854.1/ Missing YP_001119851.1/ YP_001119850.1/chromosome vietnamiensis G4 4965 5582 629 1728 4311 uid58075Calditerrivibrio 2095970 2108863 YP_004052086.1/ YP_004052085.1/YP_004052084.1/ YP_004052081.1/ YP_004052080.1/ YP_004052079.1/ Splitchromosome nitroreducens DSM 4966 5583 630 2779 1729 4312 methylase.19672 uid60821 YP_004052083.1/ 2780 Carboxydothermus 2348825 2361903YP_361448.1/ YP_361447.1/ YP_361446.1/ YP_361445.1/ YP_361444.1/YP_361443.1/ chromosome hydrogenoformans Z 4967 5584 631 2781 1730 43132901 uid57821 Chlorobium 1811542 1826075 YP_001960082.1/ YP_001960081.1/YP_001960080.1/ YP_001960077.1/ YP_001960076.1/ YP_001960075.1/chromosome phaeobacteroides BS1 4968 5585 632 2782 1731 4314 uid58131Clostridium ljungdahlii 3566681 3580351 YP_003781421.1/ YP_003781420.1/YP_003781419.1/ YP_003781418.1/ YP_003781417.1/ YP_003781415.1/chromosome DSM 13528 uid50583 4971 5588 635 2786 1734 4317 Clostridium3014510 3029581 YP_003822961.1/ YP_003822960.1/ YP_003822959.1/YP_003822957.1/ YP_003822954.1/ YP_003822953.1/ chromosomesaccharolyticum WM1 4972 5589 636 2787 1735 4318 uid51419 Clostridium3993474 4020337 YP_003823839.1/ YP_003823838.1/ YP_003823837.1/YP_003823836.1/ YP_003823832.1/ YP_003823831.1/ chromosomesaccharolyticum WM1 4973 5590 637 2788 1736 4319 uid51419 Clostridiumsticklandii 1172754 1188586 YP_003936059.1/ YP_003936060.1/YP_003936061.1/ YP_003936062.1/ YP_003936065.1/ YP_003936066.1/chromosome DSM 519 uid59585 4974 5591 638 2790 1737 4320 ClostridiumSY8519 1975332 1994112 YP_004708906.1/ YP_004708907.1/ YP_004708908.1/YP_004708910.1/ YP_004708913.1/ YP_004708914.1/ chromosome uid68705 49755592 639 2791 1738 4321 Cupriavidus necator N 1 3467559 3485436YP_004687115.1/ YP_004687114.1/ YP_004687113.1/ YP_004687110.1/YP_004687108.1/ YP_004687107.1/ chromosome uid68689 4976 5593 640 27921739 4322 Cyanothece PCC 8802 2210340 2225482 Missing YP_003137927.1/YP_003137926.1/ YP_003137924.1/ YP_003137920.1/ YP_003137919.1/ Onechromosome uid59143 5594 641 2794 1740 4323 complete YP_003137923.1/methylase 2795 and one truncated. Dehalococcoides VS 244220 257903YP_003329752.1/ YP_003329751.1/ YP_003329750.1/ YP_003329749.1/YP_003329748.1/ chromosome uid42393 4977 642 2795 1741 4324Dehalogenimonas 1589660 1603624 YP_003759227.1/ YP_003759226.1/YP_003759225.1/ YP_003759224.1/ YP_003759223.1/ YP_003759222.1/chromosome lykanthroporepellens BL 4978 5595 643 2796 1742 4325 DC 9uid48131 Desulfitobacterium 792814 806836 YP_516908.1/ YP_516909.1/YP_516910.1/ YP_516912.1/ YP_516913.1/ chromosome hafniense Y51 uid586054979 644 2797 1743 4326 Desulfomicrobium 1457033 1471353 YP_003157843.1/YP_003157842.1/ YP_003157841.1/ YP_003157840.1/ YP_003157838.1/YP_003157837.1/ chromosome baculatum DSM 4028 4981 5597 646 2799 17454328 uid59217 Desulfovibrio 4090474 4104148 YP_002954994.1/YP_002954993.1/ YP_002954992.1/ YP_002954991.1/ YP_002954989.1/YP_002954988.1/ chromosome magneticus RS 1 4983 5599 648 2801 1747 4330uid59309 Desulfovibrio vulgaris 2096179 2110453 Missing YP_011241.1/YP_011240.1/ YP_011237.1/ YP_011235.1/ YP_011234.1/ chromosomeHildenborough uid57645 5600 649 2802 1748 4331 Desulfurivibrio 25129362529324 YP_003691455.1/ YP_003691454.1/ YP_003691453.1/ YP_003691451.1/YP_003691448.1/ YP_003691447.1/ chromosome alkaliphilus AHT2 4985 5602651 2804 1750 4333 uid49487 Erwinia pyrifoliae Ep1 3075240 3088692YP_002649786.1/ YP_002649787.1/ YP_002649788.1/ YP_002649789.1/YP_002649790.1/ YP_002649791.1/ chromosome 96 uid40659 4989 5606 6552809 1754 4337 Erythrobacter litoralis 2963192 2977694 YP_459832.1/YP_459834.1/ YP_459835.1/ YP_459838.1/ YP_459839.1/ YP_459840.1/chromosome HTCC2594 uid58299 4990 5607 656 2810 1755 4338 Escherichiacoli HS 335287 354281 YP_001457107.1/ YP_001457108.1/ YP_001457109.1/YP_001457110.1/ YP_001457111.1/ YP_001457112.1/ chromosome uid58393 49935610 659 2814 1758 4341 Escherichia coli O111 H 5250834 5264277YP_003237428.1/ YP_003237427.1/ YP_003237426.1/ YP_003237425.1/YP_003237424.1/ YP_003237423.1/ chromosome 11128 uid41023 4994 5611 6602815 1759 4342 Escherichia fergusonii 13818 28889 YP_002394569.1/YP_002394570.1/ YP_002394571.1/ YP_002394573.1/ YP_002394574.1/YP_002394575.1/ plasmid ATCC 35469 uid59375 4995 5612 661 2816 1760 4343Exiguobacterium 325862 338926 YP_001812815.1/ YP_001812816.1/YP_001812817.1/ YP_001812818.1/ YP_001812819.1/ YP_001812820.1/chromosome sibiricum 255 15 4996 5613 662 2817 1761 4344 uid58053Gallionella 927586 949287 YP_003846678.1/ YP_003846679.1/YP_003846680.1/ YP_003846683.1/ YP_003846691.1/ YP_003846692.1/chromosome capsiferriformans ES 2 4998 5615 664 2819 1763 4346 uid51505Gallionella 1651741 1666042 YP_003847303.1/ YP_003847304.1/YP_003847305.1/ YP_003847308.1/ YP_003847309.1/ YP_003847310.1/chromosome capsiferriformans ES 2 4999 5616 665 2820 1764 4347 uid51505Geobacillus WCH70 1353239 1364077 YP_002949411.1/ YP_002949412.1/YP_002949413.1/ YP_002949414.1/ YP_002949415.1/ Missing chromosomeuid59045 5000 5617 666 2821 1765 Geobacter sulfurreducens 23165762330995 NP_953159.2/ NP_953158.1/ NP_953157.1/ NP_953156.1/ NP_953155.1/NP_953154.1/ chromosome PCA uid57743 5001 5618 667 2822 1766 4348Haliscomenobacter 16081 32343 YP_004451379.1/ YP_004451378.1/YP_004451377.1/ YP_004451376.1/ YP_004451373.1/ YP_004451372.1/ plasmidhydrossis DSM 1100 5002 5619 668 2823 1767 4349 uid66777 Lactobacilluscasei 2008786 2025228 YP_003789119.1/ YP_003789118.1/ YP_003789117.1/YP_003789114.1/ YP_003789113.1/ YP_003789112.1/ One chromosome Zhanguid50673 5007 5624 673 2828 1772 4351 complete YP_003789116.1/ methylase2829 and one truncated Lactobacillus johnsonii 939272 953168YP_003293166.1/ YP_003293165.1/ YP_003293164.1/ YP_003293163.1/YP_003293159.1/ Missing chromosome FI9785 uid41735 5010 5627 676 28321775 Lactobacillus reuteri 1310070 1322351 YP_004649803.1/YP_004649804.1/ YP_004649805.1/ YP_004649806.1/ YP_004649809.1/ Missingchromosome SD2112 uid55357 5011 5628 677 2833 1776 Lactobacillusrhamnosus 2154005 2170387 YP_003171846.1/ YP_003171845.1/YP_003171844.1/ YP_003171841.1/ YP_003171840.1/ YP_003171839.1/ Onechromosome GG uid59313 5013 5630 679 2836 1778 4353 completeYP_003171843.1/ methylase 2837 and one truncated. Leuconostoc kimchii34362 50836 YP_003620562.1/ YP_003620561.1/ YP_003620560.1/YP_003620557.1/ YP_003620556.1/ YP_003620555.1/ One chromosome IMSNU11154 uid48589 5014 5631 680 2838 1779 4354 complete YP_003620559.1/methylase 2839 and one truncated. Magnetospirillum 2215526 2231932YP_421401.1/ YP_421402.1/ YP_421403.1/ YP_421407.1/ YP_421409.1/YP_421410.1/ chromosome magneticum AMB 1 5060 5682 731 2912 1830 4398uid58527 YP_421406.1/ 732 Marinobacter aquaeolei 627682 647741YP_957843.1/ YP_957844.1/ YP_957845.1/ YP_957849.1/ YP_957852.1/YP_957853.1/ chromosome VT8 uid59419 5061 5683 733 2913 1831 4399Methanobrevibacter 1795596 1818644 YP_001274322.1/ YP_001274323.1/YP_001274324.1/ YP_001274316.1/ YP_001274326.1/ YP_001274327.1/ Onechromosome smithii ATCC 35061 5015 5632 681 2840 1780 6165 completeuid58827 YP_001274317.1/ methylase 2841 and 5 YP_001274318.1/ truncated.2842 YP_001274319.1/ 2843 YP_001274320.1/ 2844 YP_001274321.1/ 2845Methanosarcina 2919156 2939722 Missing NP_617281.1/ NP_617280.1/NP_617279.1/ NP_617273.1/ NP_617272.1/ chromosome acetivorans C2A 5636685 2852 1784 4358 uid57879 Methanosarcina mazei 193892 214864 MissingNP_632177.1/ NP_632178.1/ NP_632180.1/ NP_632187.1/ NP_632188.1/chromosome Gol uid57893 5637 686 2853 1785 4359 Methanospirillum 12138181228530 YP_502553.1/ YP_502552.1/ YP_502551.1/ YP_502549.1/ YP_502546.1/YP_502545.1/ chromosome hungatei JF 1 uid58181 5017 5638 687 2854 17864360 Methanospirillum 2137148 2153998 Missing YP_503327.1/ YP_503328.1/YP_503329.1/ YP_503337.1/ YP_503338.1/ chromosome hungatei JF 1 uid581815639 688 2855 1787 4361 Microlunatus 4106044 4121697 YP_004574223.1/YP_004574224.1/ YP_004574225.1/ YP_004574226.1/ YP_004574231.1/YP_004574232.1/ methylase chromosome phosphovorus NM 1 5018 5640 6892856 1788 4362 split into uid68055 YP_004574227.1/ three. 2857YP_004574229.1/ 2858 Moorella thermoacetica 2331992 2347051 YP_431074.1/YP_431073.1/ YP_431072.1/ YP_431068.1 YP_431067.1/ YP_431066.1/ Splitchromosome ATCC 39073 uid58051 5019 5641 690 YP_431071.1 1789 4363methylase. Nostoc punctiforme PCC 6986741 7000722 YP_001868910.1/YP_001868903.1/ YP_001868904.1/ YP_001868905.1/ YP_001868908.1/YP_001868909.1/ chromosome 73102 uid57767 5062 5684 734 2914 1832 4400Parvularcula 1068561 1081391 YP_003854347.1/ YP_003854346.1/YP_003854345.1/ YP_003854344.1/ YP_003854343.1/ YP_003854342.1/chromosome bermudensis HTCC2503 5020 5642 691 2861 1790 4364 uid51641Pelobacter propionicus 2459986 2475529 YP_901956.1/ YP_901955.1/YP_901954.1/ YP_901953.1/ YP_901949.1/ YP_901948.1/ chromosome DSM 2379uid58255 5022 5644 693 2864 1792 4366 Pelodictyon 2122302 2136557YP_002018860.1/ YP_002018861.1/ YP_002018862.1/ YP_002018863.1/YP_002018865.1/ YP_002018866.1/ chromosome phaeoclathratiforme BU 50235645 694 2865 1793 4367 1 uid58173 Photorhabdus 470261 481491YP_003039266.1/ YP_003039267.1/ YP_003039268.1/ YP_003039269.1/YP_003039270.1/ Missing chromosome asymbiotica ATCC 5024 5646 695 28661794 43949 uid59243 Polaromonas JS666 120587 142415 YP_551792.1/YP_551793.1/ YP_551794.1/ YP_551797.1/ YP_551801.1/ YP_551802.1/ plasmiduid58207 5025 5647 696 2867 1795 4368 Pseudomonas 940354 955543YP_004351894.1/ YP_004351895.1/ YP_004351896.1/ YP_004351897.1/YP_004351899.1/ YP_004351900.1/ chromosome brassicacearum NFM421 50265648 697 2868 1796 4369 uid66303 Psychrobacter 1973414 1984296YP_580872.1/ YP_580871.1/ YP_580870.1/ YP_580869.1/ YP_580868.1/ Missingchromosome cryohalolentis K5 5028 5650 699 2871 1798 uid58373Rhodobacter sphaeroides 628478 642432 YP_001169907.1/ YP_001169906.1/Rsph17025_3734/ YP_001169903.1/ YP_001169902.1/ YP_001169901.1/ plasmidATCC 17025 uid58451 5063 5685 615 2915 1833 4401 Rhodococcus 30991103111990 YP_002766345.1/ YP_002766344.1/ YP_002766343.1/ YP_002766342.1/YP_002766341.1/ YP_002766340.1/ chromosome erythropolis PR4 5029 5651700 2872 1799 4371 uid59019 Rhodopseudomonas 1186657 1200147YP_001990139.1/ YP_001990140.1/ YP_001990141.1/ YP_001990142.1/YP_001990143.1/ YP_001990144.1/ chromosome palustris TIE 1 uid58995 50305652 701 2873 1800 4372 Runella slithyformis 4922401 4938169YP_004657746.1/ YP_004657745.1/ YP_004657744.1/ YP_004657743.1/YP_004657738.1/ YP_004657737.1/ chromosome DSM 19594 uid68317 5064 5686735 2916 1834 4402 Saccharophagus 3482582 3496946 YP_528231.1/YP_528230.1/ YP_528229.1/ YP_528228.1/ YP_528225.1/ YP_528224.1/chromosome degradans 2 40 uid57921 5031 5653 702 2874 1801 4373Salmonella enterica 4736680 4751839 NP_463357.1/ NP_463356.1/NP_463355.1/ NP_463354.1/ NP_463351.1/ NP_463350.1/ chromosome serovarTyphimurium 5034 5656 705 2877 1804 4376 LT2 uid57799 Selenomonassputigena 820667 833330 YP_004413153.1/ YP_004413154.1/ YP_004413155.1/YP_004413156.1/ YP_004413158.1/ Missing chromosome ATCC 35185 uid663355040 5662 711 2883 1810 Shewanella ANA 3 2138213 2155059 YP_869455.1/YP_869456.1/ YP_869457.1/ YP_869458.1/ YP_869460.1/ Missing chromosomeuid58347 5041 5663 712 2884 1811 Shewanella MR 4 2013300 2030132YP_733838.1/ YP_733839.1/ YP_733840.1/ YP_733841.1/ YP_733843.1/ Missingchromosome uid58345 5042 5664 713 2885 1812 Slackia 349611 366195YP_003142715.1/ YP_003142714.1/ YP_003142713.1/ YP_003142710.1/YP_003142712.1/ YP_003142708.1/ Two full chromosome heliotrinireducensDSM 5043 5665 714 2886 1813 4382 length 20476 uid59051 YP_003142711.1/methylases. 2887 Spirosoma linguale DSM 5893753 5908211 YP_003389619.1/YP_003389620.1/ YP_003389621.1/ YP_003389624.1/ YP_003389625.1/YP_003389626.1/ chromosome 74 uid43413 5044 5666 715 2888 1814 4383Sulfuricurvum kujiense 1691724 1708882 YP_004060547.1/ YP_004060548.1/YP_004060549.1/ YP_004060550.1 YP_004060557.1/ YP_004060558.1/ Splitchromosome DSM 16994 uid60789 5045 5667 716 YP_004060555.1 1815 4384methylase. Syntrophomonas wolfei 2823947 2838578 YP_755156.1/YP_755155.1/ YP_755154.1/ YP_755151.1 YP_755149.1/ YP_755148.1/ Onechromosome Goettingen uid58179 5048 5670 719 YP_755153.1 1818 4387complete methylase and one truncated. Syntrophomonas wolfei 11196271137079 YP_753673.1/ YP_753674.1/ YP_753675.1/ YP_753676.1/ YP_753683.1/YP_753684.1/ Methylase chromosome Goettingen uid58179 5047 5669 718 28931817 4386 split into YP_753679.1/ three. 2894 YP_753682.1/ 2895Syntrophus 932423 951906 YP_460955.1/ YP_460954.1/ YP_460953.1/YP_460949.1/ YP_460946.1/ YP_460945.1/ chromosome aciditrophicus SB 50495671 720 2899 1819 4388 uid58539 Tepidanaerobacter Re1 564518 579372YP_004460026.1/ YP_004460027.1/ YP_004460028.1/ YP_004460030.1/YP_004460031.1/ YP_004460032.1/ chromosome uid66873 5051 5673 722 29021821 4390 Thauera MZ1T uid58987 329333 347549 YP_002353978.1/YP_002353979.1/ YP_002353980.1/ YP_002353983.1/ YP_002353988.1/YP_002353989.1/ chromosome 5052 5674 723 2903 1822 4391Thermoanaerobacterium 483298 496318 YP_003851149.1/ YP_003851150.1/YP_003851151.1/ YP_003851152.1/ YP_003851153.1/ YP_003851154.1/chromosome thermosaccharolyticum 5054 5676 725 2905 1824 4393 DSM 571uid51639 Vibrio cholerae MJ 1236 3021296 3036340 YP_002879448.1/YP_002879447.1/ YP_002879446.1/ YP_002879444.1/ YP_002879443.1/YP_002879442.1/ chromosome uid59387 5057 5679 728 2909 1827 4395Zymomonas mobilis 1590155 1602970 YP_003226496.1/ YP_003226497.1/YP_003226498.1/ YP_003226499.1/ YP_003226500.1/ YP_003226501.1/chromosome NCIMB 11163 uid41019 5059 5681 730 2911 1829 4397Thioalkalivibrio sp. 1195163 1212198 YP_003460374.1/ YP_003460375.1/TK90_1129 (no YP_003460378.1/ YP_003460381.1/ YP_003460382.1/ chromosomeK90mix 6225 6227 accession)/ 6231 6233 6235 6229 *Numbers are presentedby Accession NO./SEQ ID NO.

TABLE 3 BREX type 5 systems Genomic Genomic Start End Organism PointPoint BrxA* BrxB* BrxC/PglY* PglX* Haloarcula 401474 426563YP_004786064.1/ YP_004786062.1/ YP_004786061.1/ YP_004786055.1/hispanica 5068 5691 744 2922 ATCC 33960 YP_004786063.1/ YP_004786058.1/uid72475 745 2923 Halobacterium 213889 239329 YP_001690762.1/YP_001690760.1/ YP_001690761.1/ YP_001690755.1/ salinarum R1 5066 5689741 2919 uid61571 YP_001690759.1/ 740 halophilic 245839 265731YP_004809887.1/ YP_004809889.1/ YP_004809888.1/ YP_004809893.1/ archaeon5069 5692 746 2924 DL31 YP_004809890.1/ uid72619 747 Halopiger 275443305939 YP_004595482.1/ YP_004595484.1/ YP_004595483.1/ YP_004595494.1/xanaduensis 5065 5687 736 2917 SH 6 YP_004595485.1/ uid68105 737Halorubrum 421884 442192 YP_002564617.1/ YP_002564615.1/ YP_002564616.1/YP_002564611.1/ lacusprofundi 5067 5690 743 2920 ATCC 49239YP_002564614.1/ uid58807 742 Halorhabdus 1919731 1943689 257052977/YP_003130812.1/ YP_003130811.1/ YP_003130818.1/ utahensis 6171 5688 7382918 DSM 12940 YP_003130813.1/ uid59189 739 Methylase Replicon OrganismPglZ* BrxHII* State Type Haloarcula YP_004786056.1/ YP_004786054.1/ Twofull chromosome hispanica 1839 3501 length ATCC 33960 methylases.uid72475 Halobacterium YP_001690753.1/ plasmid salinarum R1 1837uid61571 halophilic YP_004809895.1/ YP_004809896.1/ plasmid archaeon1840 3502 DL31 uid72619 Halopiger YP_004595497.1/ YP_004595499.1/chromosome xanaduensis 1835 3498 SH 6 uid68105 HalorubrumYP_002564610.1/ YP_002564609.1/ chromosome lacusprofundi 1838 3500 ATCC49239 uid58807 Halorhabdus YP_003130820.1/ YP_003130822.1/ chromosomeutahensis 1836 3499 DSM 12940 uid59189 *Numbers are presented byAccession NO./SEQ ID NO.

TABLE 4 BREX type 6 systems Genomic Genomic Start End Organism PointPoint BrxE* BrxA* BrxB* BrxC/PglY* Anaeromyxobacter 1284321 1301040YP_002491563.1/ YP_002491564.1/ YP_002491565.1/ YP_002491566.1/dehalogenans 2CP 6038 5072 5695 751 1 uid58989 Haliangium 16113131628687 YP_003265686.1/ YP_003265687.1/ YP_003265688.1/ YP_003265689.1/ochraceum DSM 6040 5074 5697 753 14365 uid41425 Haliangium 798493 815906YP_003265127.1/ YP_003265128.1/ YP_003265129.1/ YP_003265130.1/ochraceum DSM 6039 5073 5696 752 14365 uid41425 Planctomyces 39793043995634 YP_003631101.1/ YP_003631100.1/ YP_003631099.1/ YP_003631098.1/limnophilus DSM 6037 5071 5694 750 3776 uid48643 Organism PglX* PglZ*BrxD* BrxHI* Anaeromyxobacter YP_002491567.1/ YP_002491568.1/YP_002491570.1/ YP_002491571.1/ dehalogenans 2CP 2928 1843 4442 3623 1uid58989 Haliangium YP_003265690.1/ YP_003265691.1/ YP_003265692.1/YP_003265693.1/ ochraceum DSM 2930 1845 4444 3625 14365 uid41425Haliangium YP_003265131.1/ YP_003265132.1/ YP_003265133.1/YP_003265134.1/ ochraceum DSM 2929 1844 4443 3624 14365 uid41425Planctomyces YP_003631097.1/ YP_003631096.1/ YP_003631095.1/YP_003631094.1/ limnophilus DSM 2927 1842 4441 3622 3776 uid48643*Numbers are presented by Accession NO./SEQ ID NO.

TABLE 5 BREX type 3 systems Genomic Genomic Start End Organism PointPoint BrxF* BrxC/PglY* PglXI* BrxHII* PglZ* BrxA* Acidothermus 895934911157 YP_872570.1/ YP_872571.1/ YP_872573.1/ YP_872575.1/ YP_872576.1/YP_872577.1/ cellulolyticus 11B 5980 755 3344 3504 1847 5076 uid58501Parvibaculum 1304574 1320948 YP_001412459.1/ YP_001412458.1/YP_001412455.1/ YP_001412454.1/ YP_001412453.1/ YP_001412452.1/lavamentivorans DS 1 5992 769 3360 3515 1860 5089 uid58739 Parvibaculum3796620 3812997 YP_001414809.1/ YP_001414810.1/ YP_001414813.1/YP_001414814.1/ YP_001414815.1/ YP_001414816.1/ lavamentivorans DS 15993 770 3361 3516 1861 5090 uid58739 Chloroflexus aggregans 13762561391910 YP_002462464.1/ YP_002462466.1/ YP_002462465.1/ YP_002462469.1/YP_002462470.1/ YP_002462471.1/ DSM 9485 uid58621 5983 759 3348 35071851 5080 Desulfovibrio 1822100 1838149 YP_004121411.1/ YP_004121412.1/YP_004121414.1/ YP_004121416.1/ YP_004121417.1/ YP_004121418.1/aespoeensis Aspo 2 5984 760 3350 3508 1852 5081 uid42613 Methanosalsumzhilinae 1398870 1421705 YP_004616377.1/ YP_004616376.1/ YP_004616375.1/YP_004616371.1/ YP_004616370.1/ YP_004616369.1/ DSM 4017 uid68249 5987764 3353 3510 1855 5084 Caldicellulosiruptor 671655 683698YP_004025779.1/ YP_004025780.1/ YP_004025781.1/ Calkr_0625/YP_004025782.1/ YP_004025783.1/ kristjanssonii 177R1B 5982 758 3347 61731850 5079 uid60393 Pelotomaculum 698030 719373 YP_001211256.1/YP_001211265.1/ YP_001211266.1/ YP_001211267.1/ / thermopropionicum SI616 3403 3493 1715 uid58877 Thermoanaerobacter 974096 986151YP_004185913.1/ YP_004185914.1/ YP_004185915.1/ YP_004185916.1/YP_004185917.1/ YP_004185918.1/ brockii finnii Ako 1 5999 777 3367 35191867 5096 uid55639 Thermoanaerobacter 981903 993958 YP_001664916.1/YP_001664917.1/ YP_001664918.1/ YP_001664919.1/ YP_001664920.1/YP_001664921.1/ pseudethanolicus ATCC 6001 779 3369 3520 1869 5098 33223uid58339 Thermoanaerobacterium 1036830 1048931 YP_004470679.1/YP_004470680.1/ YP_004470681.1/ YP_004470682.1/ YP_004470683.1/YP_004470684.1/ xylanolyticum LX 11 6004 782 3372 3523 1872 5101uid63163 Thermoanaerobacter 1369768 1379419 YP_003477173.1/YP_003477172.1/ YP_003477171.1/ Missing YP_003477169.1/ YP_003477168.1/italicus Ab9 uid46241 6000 778 3368 1868 5097 Syntrophothermus 13074171316966 YP_003702612.1/ YP_003702611.1/ YP_003702610.1/ MissingYP_003702609.1/ YP_003702608.1/ lipocalidus DSM 12680 5995 772 3363 18635092 uid49527 Acetohalobium 1465881 1481230 YP_003827965.1/YP_003827964.1/ YP_003827963.1/ Missing YP_003827962.1/ YP_003827961/arabaticum DSM 5501 5979 754 3343 1846 5075 uid51423 Dichelobacternodosus 185804 200163 YP_001209111.1/ YP_001209110.1/ YP_001209109.1/YP_001209106.1/ YP_001209105.1/ YP_001209104.1/ VCS1703A uid57643 5985761 3351 3509 1853 5082 Nitrosococcus oceani 53559 70307 YP_342127.1/YP_342128.1/ YP_342131.1/ YP_342135.1/ YP_342136.1/ YP_342137.1/ ATCC19707 uid58403 5990 767 3358 3513 1858 5087 Nitrosococcus watsonii 4370358760 YP_003759356.1/ YP_003759357.1/ YP_003759359.1/ YP_003759363.1/YP_003759364.1/ YP_003759365.1/ C 113 uid50331 5991 768 3359 3514 18595088 Methylacidiphilum 317802 333912 YP_001938984.1/ YP_001938983.1/YP_001938982.1/ YP_001938980.1/ YP_001938979.1/ YP_001938978.1/infernorum V4 5988 765 3356 3511 1856 5085 uid59161 Thermanaerovibrio1551826 1568220 YP_003318004.1/ YP_003318003.1/ YP_003318001.1/YP_003317999.1/ YP.003317998.1/ YP_003317997.1/ acidaminovorans DSM 5998776 3366 3518 1866 5095 6589 uid41925 Planctomyces 1319343 1335543YP_004268781.1/ YP_004268780.1/ YP_004268778.1/ YP_004268777.1/YP_004268776.1/ YP_004268775.1/ brasiliensis DSM 5305 5994 771 3362 35171862 5091 uid60583 Tepidanaerobacter Re1 1934428 1943118 YP_004461309.1/YP_004461308.1/ YP_004461307.1/ missing YP_004461306.1/ YP_004461305.1/uid66873 5997 775 3365 1865 5096 *Numbers are presented by AccessionNO./SEQ ID NO.

TABLE 6 BREX type 2 systems Genomic Genomic Start End Organism PointPoint PglW* PglX* PglY* PglZ* BrxD* BrxHI* Candidatus 3869550 3890263YP_003168573.1/ YP_003168572.1/ YP_003168567.1/ YP_003168566.1/YP_003168565.1/ YP_003168564.1/ Accumulibacter 6092 2932 784 1874 44453596 phosphatis clade IIA UW 1 uid59207 Corynebacterium 1913373 1933608YP_004760098.1/ YP_004760099.1/ YP_004760100.1/ YP_004760101.1/YP_004760102.1/ YP_004760103.1/ variabile DSM 44702 6094 2934 787 18764446 3598 uid62003 Frankia CcI3 3489708 3507585 YP_482039.1/YP_482040.1/ YP_482041.1/ YP_482042.1/ YP_482043.1/ YP_482044.1/uid58397 6095 2935 788 1877 4447 3599 Frankia EuI1c 6951904 6971263YP_004019519.1/ YP_004019520.1/ YP_004019521.1/ YP_004019522.1/YP_004019523.1/ YP_004019524.1/ uid42615 6096 2936 789 1878 4448 3600Hahella chejuensis 3587257 3606877 YP_434642.1/ YP_434639.1/YP_434638.1/ YP_434637.1/ YP_434636.1/ YP_434635.1/ KCTC 2396 uid584836114 2953 806 1894 4462 3615 Haliangium 1565170 1582937 YP_003265661.1/YP_003265662.1/ YP_003265663.1/ YP_003265664.1/ YP_003265665.1/YP_003265666.1/ ochraceum DSM 6097 2937 790 1879 791 3601 14365 uid41425Microlunatus 3075705 3093432 YP_004573280.1/ YP_004573279.1/YP_004573278.1/ YP_004573277.1/ YP_004573276.1/ YP_004573275.1/phosphovorus NM 1 6098 2938 792 1880 4449 3602 uid68055 Micromonospora1329830 1350410 YP_003834429.1/ YP_003834430.1/ YP_003834433.1/YP_003834434.1/ YP_003834435.1/ YP_003834436.1/ aurantiaca ATCC 60992939 793 1881 4450 3603 27029 uid42501 Mycobacterium 3386977 3404461YP_001134469.1/ YP_001134468.1/ YP_001134467.1/ YP_001134466.1/YP_001134465.1/ YP_001134464.1/ gilvum PYR GCK 6100 2940 794 1882 44513604 uid59421 Polaromonas 170793 191771 YP_973309.1/ YP_973307.1/YP_973304.1/ YP_973303.1/ YP_973302.1/ YP_973301.1/ naphthalenivorans6102 2942 796 1884 4453 3606 CJ2 uid58273 Saccharopolyspora 57140835716377 YP_001107302.1/ YP_001107301.1/ YP_001107300.1/ YP_001107299.1/YP_001107298.1/ YP_001107297.1/ erythraea NRRL 2338 6104 2945 798 18864455 3608 uid62947 Sorangium 1.10E+07 10706714 YP_001618324.1/YP_001618325.1/ YP_001618331.1/ YP_001618334.1/ YP_001618335.1/YP_001618336.1/ cellulosum So ce 56 6107 2948 801 1889 4458 3611uid61629 Streptomyces 7348537 7376403 NP_630703.1/ NP_733709.1/NP_630711.1/ NP_630712.1/ NP_630715.1/ NP_630716.1/ coelicolor A3 2 61102949 802 1890 4459 3612 uid57801 Streptomyces griseus 1877109 1900853YP_001823112.1/ YP_001823113.1/ YP_001823118.1/ YP_001823119.1/YP_001823122.1/ YP_001823123.1/ NBRC 13350 6111 2950 803 1891 4460 3613uid58983 Thermobifida fusca 810381 830646 YP_288762.1/ Rsph17025_3734/YP_288770.1/ YP_288771.1/ YP_288772.1/ YP_288773.1/ YX uid57703 6112 615804 1892 4461 3614 Burkholderia 131918 145741 YP_440673.1/ YP_440674.1/YP_440675.1/ YP_440676.1/ Missing Missing thailandensis E264 6091 2931783 1873 uid58081 Thermobispora 1764882 779583 YP_003652152.1/YP_003652153.1/ YP_003652154.1/ YP_003652155.1/ Missing Missing bisporaDSM 43833 6113 2952 805 1893 uid48999 Saccharomonospora 508003 530821YP_003132413.1/ YP_003132422.1/ YP_003132423.1/ YP_003132424.1/YP_003132425.1/ YP_003132426.1/ viridis DSM 43017 6103 2944 797 18854454 3607 *Numbers are presented by Accession NO./SEQ ID NO.

TABLE 7 BREX type 4 systems Genomic Genomic Start End Organism PointPoint BrxP* BrxC/PglY* PglZ* BrxL* Candidatus 837185 848109YP_001716949.1/ YP_001716950.1/ YP_001716952.1/ YP_001716953.1/Desulforudis 3431 809 1897 4405 audaxviator MP104C uid59067Coprothermobacter 1373789 1384972 YP_002247820.1/ YP_002247818.1/YP_002247817.1/ YP_002247816.1/ proteolyticus DSM 3433 810 1898 44065265 uid59253 YP_002247819.1/ 3432 Denitrovibrio 583467 593589YP_003503325.1/ YP_003503326.1/ YP_003503327.1/ YP_003503328.1/acetiphilus DSM 3435 813 1900 4408 12809 uid46657 Geobacter M21 937933948070 YP_003020622.1/ YP_003020623.1/ YP_003020624.1/ YP_003020625.1/uid59037 3437 815 1902 4410 Prevotella 414128 424371 YP_004328097.1/YP_004328099.1/ YP_004328100.1/ YP_004328101.1/ denticola F0289 3438 8161903 4411 uid65091 Thermomicrobium 91162 104624 YP_002523385.1/YP_002523389.1/ YP_002523390.1/ roseum DSM 5159 614 1714 4299 uid59341Thermotoga 1747153 1756730 YP_001245328.1/ YP_001245329.1/YP_001245330.1/ YP_001245331.1/ petrophila RKU 1 3439 817 1904 4412uid58655 *Numbers are presented by Accession NO./SEQ ID NO.

TABLE 8 Summary of distribution of BREX types across genomes Taxon BREXBREX BREX BREX BREX BREX ID Organism Kingdom #1 #5 #6 #3 #2 #4 Comments1 882 Desulfovibrio vulgaris Bacteria X Hildenborough uid57645 2 56780Syntrophus aciditrophicus Bacteria X SB uid58539 3 60480 Shewanella MR 4uid58345 Bacteria X 4 62140 Acidiphilium multivorum Bacteria X AIU301uid63345 5 63737 Nostoc punctiforme PCC Bacteria X 73102 uid57767 676114 Aromatoleum aromaticum Bacteria X EbN1 uid58231 7 85643 ThaueraMZ1T uid58987 Bacteria X 8 94122 Shewanella ANA 3 Bacteria X uid58347 999287 Salmonella enterica serovar Bacteria X Typhimurium LT2 uid57799 10138119 Desulfitobacterium Bacteria X hafniense Y51 uid58605 11 188937Methanosarcina acetivorans Archaea X C2A uid57879 12 192952Methanosarcina mazei Go1 Archaea X uid57893 13 203122 Saccharophagusdegradans Bacteria X 2 40 uid57921 14 234621 Rhodococcus erythropolisBacteria X PR4 uid59019 15 243231 Geobacter sulfurreducens Bacteria XPCA uid57743 16 246194 Carboxydothermus Bacteria X hydrogenoformans Z2901 uid57821 17 262543 Exiguobacterium sibiricum Bacteria X 255 15uid58053 18 264732 Moorella thermoacetica Bacteria X ATCC 39073 uid5805119 269482 Burkholderia vietnamiensis Bacteria X G4 uid58075 20 290397Anaeromyxobacter Bacteria X dehalogenans 2CP C uid58135 21 296591Polaromonas JS666 Bacteria X uid58207 22 311424 Dehalococcoides VSBacteria X uid42393 23 314225 Erythrobacter litoralis Bacteria XHTCC2594 uid58299 24 314260 Parvularcula bermudensis Bacteria X HTCC2503uid51641 25 323259 Methanospirillum hungatei Archaea XX This genomecontains JF 1 uid58181 two BREX systems of type 1 26 324925 PelodictyonBacteria X phaeoclathratiforme BU 1 uid58173 27 331112 Escherichia coliHS Bacteria X uid58393 28 331678 Chlorobium Bacteria X phaeobacteroidesBS1 uid58131 29 335284 Psychrobacter Bacteria X cryohalolentis K5uid58373 30 335541 Syntrophomonas wolfei Bacteria XX This genomecontains Goettingen uid58179 two BREX systems of type 1 31 338966Pelobacter propionicus Bacteria X DSM 2379 uid58255 32 342108Magnetospirillum Bacteria X magneticum AMB 1 uid58527 33 349102Rhodobacter sphaeroides Bacteria X ATCC 17025 uid58451 34 351348Marinobacter aquaeolei Bacteria X VT8 uid59419 35 395494 GallionellaBacteria XX This genome contains capsiferriformans ES 2 two BREX systemsof uid51505 type 1 36 395960 Rhodopseudomonas Bacteria X palustris TIE 1uid58995 37 395962 Cyanothece PCC 8802 Bacteria X uid59143 38 420247Methanobrevibacter smithii Archaea X ATCC 35061 uid58827 39 471223Geobacillus WCH70 Bacteria X uid59045 40 471855 Slackiaheliotrinireducens Bacteria X DSM 20476 uid59051 41 491077 Lactobacillusreuteri Bacteria X SD2112 uid55357 42 498216 Lactobacillus casei ZhangBacteria X uid50673 43 499177 Clostridium sticklandii Bacteria X DSM 519uid59585 44 504472 Spirosoma linguale DSM Bacteria X 74 uid43413 45509173 Acinetobacter baumannii Bacteria X AYE uid61637 46 525897Desulfomicrobium Bacteria X baculatum DSM 4028 uid59217 47 546271Selenomonas sputigena Bacteria X ATCC 35185 uid66335 48 552811Dehalogenimonas Bacteria X lykanthroporepellens BL DC 9 uid48131 49553480 Photorhabdus asymbiotica Bacteria X ATCC 43949 uid59243 50 568703Lactobacillus rhamnosus Bacteria X GG uid59313 51 573370 Desulfovibriomagneticus Bacteria X RS 1 uid59309 52 580327 ThermoanaerobacteriumBacteria X thermosaccharolyticum DSM 571 uid51639 53 585054 Escherichiafergusonii Bacteria X ATCC 35469 uid59375 54 585396 Escherichia coliO111 H Bacteria X 11128 uid41023 55 589865 Desulfurivibrio alkaliphilusBacteria X AHT2 uid49487 56 593588 Vibrio cholerae MJ 1236 Bacteria Xuid59387 57 610130 Clostridium Bacteria XX This genome containssaccharolyticum WM1 two BREX systems of uid51419 type 1 58 622759Zymomonas mobilis Bacteria X NCIMB 11163 uid41019 59 633699Lactobacillus johnsonii Bacteria X FI9785 uid41735 60 634499 Erwiniapyrifoliae Ep1 96 Bacteria X uid40659 61 640510 Burkholderia CCGE1001Bacteria X uid42975 62 709032 Sulfuricurvum kujiense Bacteria X DSM16994 uid60789 63 743299 Acidithiobacillus Bacteria X ferrivorans SS3uid67387 64 748727 Clostridium ljungdahlii Bacteria X DSM 13528 uid5058365 760192 Haliscomenobacter Bacteria X hydrossis DSM 1100 uid66777 66761193 Runella slithyformis DSM Bacteria X 19594 uid68317 67 762051Leuconostoc kimchii Bacteria X IMSNU 11154 uid48589 68 768670Calditerrivibrio Bacteria X nitroreducens DSM 19672 uid60821 69 994484Pseudomonas Bacteria X brassicacearum NFM421 uid66303 70 999541Burkholderia gladioli BSR3 Bacteria X uid66301 71 1032480 Microlunatusphosphovorus Bacteria X X This genome contains NM 1 uid68055 two BREXsystems (types 1 and 2) 72 1042156 Clostridium SY8519 Bacteria Xuid68705 73 1042878 Cupriavidus necator N 1 Bacteria X uid68689 741209989 Tepidanaerobacter Re1 Bacteria X X This genome contains uid66873two BREX systems (types 1 and 3) 75 416348 Halorubrum lacusprofundiArchaea X ATCC 49239 uid58807 76 478009 Halobacterium salinarum ArchaeaX R1 uid61571 77 519442 Halorhabdus utahensis Archaea X DSM 12940uid59189 78 634497 Haloarcula hispanica ATCC Archaea X 33960 uid72475 79756883 halophilic archaeon DL31 Archaea X uid72619 80 797210 Halopigerxanaduensis SH Archaea X 6 uid68105 81 455488 Anaeromyxobacter BacteriaX dehalogenaris 2CP 1 uid58989 82 502025 Haliangium ochraceum BacteriaXX X This genome contains DSM 14365 uid41425 three BREX systems (twotypes 6 and one of type 2) 83 521674 Planctomyces limnophilus Bacteria XDSM 3776 uid48643 84 105559 Nitrosococcus watsonii C Bacteria X 113uid50331 85 246195 Dichelobacter nodosus Bacteria X VCS1703A uid57643 86323261 Nitrosococcus oceani Bacteria X ATCC 19707 uid58403 87 326427Chloroflexus aggregans Bacteria X DSM 9485 uid58621 88 340099Thermoanaerobacter Bacteria X pseudethanolicus ATCC 33223 uid58339 89351607 Acidothermus cellulolyticus Bacteria X 11B uid58501 90 370438Pelotomaculum Bacteria X thermopropionicum SI uid58877 91 402881Parvibaculum Bacteria XX This genome contains lavamentivorans DS 1 twoBREX systems of uid58739 type 3 92 481448 Methylacidiphilum Bacteria Xinfernorum V4 uid59161 93 509193 Thermoanaerobacter brockii Bacteria Xfinnii Ako 1 uid55639 94 525903 Thermanaerovibrio Bacteria Xacidaminovorans DSM 6589 uid41925 95 574087 Acetohalobium arabaticumBacteria X DSM 5501 uid51423 96 580331 Thermoanaerobacter Bacteria Xitalicus Ab9 uid46241 97 632335 Caldicellulosiruptor Bacteria Xkristjanssonii 177R1B uid60393 98 643562 Desulfovibrio aespoeensisBacteria X Aspo 2 uid42613 99 643648 Syntrophothermus Bacteria Xlipocalidus DSM 12680 uid49527 100 679901 Methanosalsum zhilinae ArchaeaX DSM 4017 uid68249 101 756272 Planctomyces brasiliensis Bacteria X DSM5305 uid60583 102 858215 Thermoanaerobacterium Bacteria X xylanolyticumLX 11 uid63163 103 100226 Streptomyces coelicolor A3 Bacteria X 2uid57801 104 106370 Frankia CcI3 uid58397 Bacteria X 105 269800Thermobifida fusca YX Bacteria X uid57703 106 271848 Burkholderiathailandensis Bacteria X E264 uid58081 107 298654 Frankia EuI1c uid42615Bacteria X 108 349521 Hahella chejuensis KCTC Bacteria X 2396 uid58483109 350054 Mycobacterium gilvum Bacteria X PYR GCK uid59421 110 365044Polaromonas Bacteria X naphthalenivorans CJ2 uid58273 111 405948Saccharopolyspora Bacteria X erythraea NRRL 2338 uid62947 112 448385Sorangium cellulosum So Bacteria X ce 56 uid61629 113 455632Streptomyces griseus Bacteria X NBRC 13350 uid58983 114 469371Thermobispora bispora Bacteria X DSM 43833 uid48999 115 522306Candidatus Accumulibacter Bacteria X phosphatis clade IIA UW 1 uid59207116 644283 Micromonospora aurantiaca Bacteria X ATCC 27029 uid42501 117858619 Corynebacterium variabile Bacteria X DSM 44702 uid62003 118309798 Coprothermobacter Bacteria X proteolyticus DSM 5265 uid59253 119309801 Thermomicrobium roseum Bacteria X DSM 5159 uid59341 120 390874Thermotoga petrophila Bacteria X RKU 1 uid58655 121 443144 Geobacter M21uid59037 Bacteria X 122 477974 Candidatus Desulforudis Bacteria Xaudaxviator MP104C uid59067 123 522772 Denitrovibrio acetiphilusBacteria X DSM 12809 uid46657 124 767031 Prevotella denticola F0289Bacteria X uid65091 125 396595 Thioalkalivibrio sp. Bacteria X K90mix126 471857 Saccharomonospora viridis Bacteria X DSM 43017

TABLE 10 BREX type 1 systems Genomic Genomic Start End Organism PointPoint brxA* brxB* brxC* pglX* pglZ* brxL* Acidiphilium 497516 512043326402595/ 326402594/ 326402593/ 326402591/ 326402589/ 326402588/multivorum AIU301 4953 5570 617 2766 1716 4300 uid63345 326402592/ 2767Acidithiobacillus 2361379 2377751 344200465/ 344200466/ 344200467/344200468/ 344200473/ 344200474/ ferrivorans SS3 4954 5571 618 2768 17174301 uid67387 344200470/ 2769 Acinetobacter 53839 68427 169786944/169786943/ 169786942/ 169786941/ 169786939/ 169786938/ baumannii AYE4955 5572 619 2770 1718 4302 uid61637 Alteromonas macleodii 22900172302123 407700182/ 407700181/ 407700180/ 407700179/ 407700176/ missingBlack Sea 11 4956 5573 620 2771 1719 uid176365 Anaeromyxobacter 36883933701032 86159656/ 86159655/ 86159654/ 86159653/ 86159652/ 86159651/dehalogenans 2CP C 4957 5574 621 2772 1720 4303 uid58135 Aromatoleum3091305 3115719 56478397/ 56478398/ 56478399/ 56478402/ 56478415/56478416/ aromaticum EbN1 4958 5575 622 2773 1721 4304 uid58231Arthrobacter 73164 86048 403571626/ 403571625/ 403571624/ 403571623/403571622/ 403571621/ nitroguajacolicus 4959 5576 623 2774 1722 4305Rue61a uid174511 Azospirillum lipoferum 149169 163502 374998149/374998148/ 374998147/ 374998346/ 374998144/ 374998143/ 4B uid82343 49605577 624 2775 1723 4306 Bifidobacterium 1014525 1027987 386867047/386867048/ 386867049/ 386867050/ 386867051/ 386867052/ animalis ATCC25527 4961 5578 625 2776 1724 4307 uid162513 Bordetella parapertussis553819 571957 410471294/ 410471295/ 410471296/ missing 410471301/410471302/ Bpp5 uid177516 4962 5579 626 1725 4308 Burkholderia 31192033136704 323527071/ 323527070/ 323527069/ 323527068/ 323527064/323527063/ CCGE1001 uid42975 4963 5580 627 2777 1726 4309 Burkholderiagladioli 1574842 1588059 330816346/ 330816347/ 330816348/ 330816349/330816350/ 330816351/ BSR3 uid66301 4964 5581 628 2778 1727 4310Burkholderia 2201159 2216554 134296121/ 134296120/ 134296119/ missing134296116/ 134296115/ vietnamiensis G4 4965 5582 629 1728 4311 uid58075Calditerrivibrio 2095967 2108863 313673975/ 313673974/ 313673973/313673970/ 313673969/ 313673968/ nitroreducens DSM 4966 5583 630 27791729 4312 19672 uid60821 313673972/ 2780 Carboxydothermus 23488222361903 78044585/ 78043641/ 78043274/ 78045163/ 78043267/ 78044476/hydrogenoformans Z 4967 5584 631 2781 1730 4313 2901 uid57821 Chlorobium1811539 1826075 189500612/ 189500611/ 189500610/ 189500607/ 189500606/189500605/ phaeobacteroides BS1 4968 5585 632 2782 1731 4314 uid58131Clostridium clariflavum 253005 267426 374294724/ 374294725/ 374294726/374294727/ 374294728/ 374294730/ DSM 19732 uid82345 4969 5586 633 27831732 4315 Clostridium clariflavum 3398998 3420023 374297215/ 374297214/374297213/ 374297205/ 374297210/ 374297209/ DSM 19732 uid82345 4970 5587634 2784 1733 4316 374297212/ 2785 Clostridium ljungdahlii 35666783580351 300856437/ 300856436/ 300856435/ 300856434/ 300856433/300856431/ DSM 13528 uid50583 4971 5588 635 2786 1734 4317 Clostridium3014507 3029581 302387139/ 302387138/ 302387137/ 302387135/ 302387132/302387131/ saccharolyticum WM1 4972 5589 636 2787 1735 4318 uid51419Clostridium 3993471 4020337 302388017/ 302388016/ 302388015/ 302388014/302388010/ 302388009/ saccharolyticum WM1 4973 5590 637 2788 1736 4319uid51419 302388025/ 2789 Clostridium sticklandii 1172754 1188589310658338/ 310658339/ 310658340/ 310658341/ 310658344/ 310658345/ DSM519 uid59585 4974 5591 638 2790 1737 4320 Clostridium SY8519 19753321994115 339442901/ 339442902/ 339442903/ 339442905/ 339442908/339442909/ uid68705 4975 5592 639 2791 1738 4321 Cupriavidus necator N 13467556 3485436 339327422/ 339327421/ 339327420/ 339327417/ 339327415/339327414/ uid68689 4976 5593 640 2792 1739 4322 Cyanothece PCC 88022210337 2225482 missing 257060039/ 257060038/ 257060035/ 257060032/257060031/ uid59143 5594 641 2793 1740 4323 257060036/ 2794Dehalococcoides VS 231698 257903 270307694/ missing 270307693/270307692/ 270307691/ 270307690/ uid42393 4977 642 2795 1741 4324Dehalogenimonas 1578285 1603624 300088705/ 300088704/ 300088703/300088702/ 300088701/ 300088700/ lykanthroporepellens 4978 5595 643 27961742 4325 BL DC 9 uid48131 Desulfitobacterium 783463 806836 89893421/missing 89893422/ 89893423/ 89893425/ 89893426/ hafniense Y51 4979 6442797 1743 4326 uid58605 Desulfobacula toluolica 3358272 3374466408420315/ 408420314/ 408420313/ 408420312/ 408420308/ 408420307/ Tol2uid175777 4980 5596 645 2798 1744 4327 Desulfomicrobium 1457030 1471353256829115/ 256829114/ 256829113/ 256829112/ 256829110/ 256829109/baculatum DSM 4028 4981 5597 646 2799 1745 4328 uid59217Desulfosporosinus 360901 375372 402570959/ 402570960/ 402570961/402570962/ 402570964/ 402570965/ meridiei DSM 13257 4982 5598 647 28001746 4329 uid75097 Desulfovibrio 4071986 4104148 239908253/ 239908252/239908251/ 239908250/ 239908248/ 239908247/ magneticus RS 1 4983 5599648 2801 1747 4330 uid59309 Desulfovibrio vulgaris 2096176 2110453missing 46580433/ 46580432/ 46580429/ 46580427/ 46580426/ Hildenboroughuid57645 5600 649 2802 1748 4331 Desulfovibrio vulgaris 1472132 1487041387153153/ 387153154/ 387153155/ 387153158/ 387153160/ 387153161/ RCH1uid161961 4984 5601 650 2803 1749 4332 Desulfurivibrio 2512933 2529324297570111/ 297570110/ 297570109/ 297570107/ 297570104/ 297570103/alkaliphilus AHT2 4985 5602 651 2804 1750 4333 uid49487 Enterobactercloacae 567010 586287 401762044/ 401762043/ 401762042/ 401762037/401762036/ 401762035/ ENHKU01 uid172463 4986 5603 652 2805 1751 4334401762040/ 2806 Erwinia Ejp617 2111476 2142932 385787391/ 385787390/385787389/ 385787388/ 385787387/ 385787386/ uid159955 4987 5604 653 28071752 4335 Erwinia pyrifoliae DSM 3075305 3107294 387872410/ 387872411/387872412/ 387872413/ 387872414/ 387872415/ 12163 uid159693 4988 5605654 2808 1753 4336 Erwinia pyrifoliae Ep1 3075240 3107369 259909430/259909431/ 259909432/ 259909433/ 259909434/ 259909435/ 96 uid40659 49895606 655 2809 1754 4337 Erythrobacter litoralis 2963192 297769785375770/ 85375772/ 85375773/ 85375776/ 85375777/ 85375778/ HTCC2594uid58299 4990 5607 656 2810 1755 4338 Escherichia coli clone 48883414901877 386637210/ 386637211/ 386637212/ 386637213/ 386637214/386637215/ D i14 uid162049 4991 5608 657 2811 1756 4339 Escherichia coliclone 4888341 4901877 386632290/ 386632291/ 386632292/ 386632293/386632294/ 386632295/ D i2 uid162047 4992 5609 658 2812 1757 4340Escherichia coli HS 335287 354284 157159789/ 157159790/ 157159791/157159784/ 157159793/ 157159794/ uid58393 4993 5610 659 2813 1758 4341157159792/ 2814 Escherichia coli O111 5250831 5264277 260871026/260871025/ 260871024/ 260871023/ 260871022/ 260871021/ H 11128 uid410234994 5611 660 2815 1759 4342 Escherichia fergusonii 13818 28892218561657/ 218561658/ 218561659/ 218561661/ 218561662/ 218561663/ ATCC35469 uid59375 4995 5612 661 2816 1760 4343 Exiguobacterium 325862338929 172056355/ 172056356/ 172056357/ 172056358/ 172056359/ 172056360/sibiricum 255 15 4996 5613 662 2817 1761 4344 uid58053 Flavobacterium1185032 1201974 347535923/ 347535922/ 347535921/ 347535920/ 347535914/347535913/ branchiophilum FL 15 4997 5614 663 2818 1762 4345 uid73421Gallionella 927586 949290 302878114/ 302878115/ 302878116/ 302878119/302878127/ 302878128/ capsiferriformans ES 2 4998 5615 664 2819 17634346 uid51505 Gallionella 1651741 1666045 302878739/ 302878740/302878741/ 302878744/ 302878745/ 302878746/ capsifeniformans ES 2 49995616 665 2820 1764 4347 uid51505 Geobacillus WCH70 1353239 1367357239826787/ 239826788/ 239826789/ 239826790/ 239826791/ missing uid590455000 5617 666 2821 1765 Geobacter 2316569 2330889 400756604/ 39997207/39997206/ 39997205/ 39997204/ 39997203/ sulfurreducens PCA 5001 5618 6672822 1766 4348 uid57743 Haliscomenobacter 16078 32343 332661910/332661909/ 332661908/ 332661907/ 332661904/ 332661903/ hydrossis DSM1100 5002 5619 668 2823 1767 4349 uid66777 Halobacillus halophilus4050129 4064174 386716369/ 386716368/ 386716367/ 386716366/ 386716365/missing DSM 2266 uid162033 5003 5620 669 2824 1768 Halobacteroides1069387 1096994 435853824/ 435853825/ 435853826/ 435853813/ 435853828/missing halobius DSM 5150 5004 5621 670 2825 1769 uid184862 435853827/2826 Klebsiella oxytoca 604335 617733 397655648/ 397655647/ 397655646/missing 397655645/ 397655644/ E718 uid170256 5005 5622 671 1770 4350Lactobacillus 1026146 1040270 385817604/ 385817605/ 385817606/385817607/ 385817611/ missing amylovorus GRL1118 5006 5623 672 2827 1771uid160233 Lactobacillus casei 2008783 2025228 301067096/ 301067095/301067094/ 301067091/ 301067090/ 301067089/ Zhang uid50673 5007 5624 6732828 1772 4351 301067093/ 2829 Lactobacillus helveticus 1063828 1077153385813809/ 385813808/ 385813807/ 385813806/ 385833804/ missing H10uid162017 5008 5625 674 2830 1773 Lactobacillus helveticus 10537001068738 403515037/ 403515036/ 403515035/ 403515034/ 403515031/ missingR0052 uid174439 5009 5626 675 2831 1774 Lactobacillus johnsonii 939269953168 268319510/ 268319509/ 268319508/ 268319507/ 268319503/ missingFI9785 uid41735 5010 5627 676 2832 1775 Lactobacillus reuteri 13100701322351 338203658/ 338203659/ 338203660/ 338203661/ 338203664/ missingSD2112 uid55357 5011 5628 677 2833 1776 Lactobacillus 2148408 2164793385828739/ 385828738/ 385828737/ 385828734/ 385828733/ 385828732/rhamnosus GG 5012 5629 678 2834 1777 4352 uid161983 385828736/ 2835Lactobacillus 2154002 2170387 258509095/ 258509094/ 258509093/258509090/ 258509089/ 258509088/ rhamnosus GG 5013 5630 679 2836 17784353 uid59313 258509092/ 2837 Leuconostoc kimchii 34359 50836 296110181/296110180/ 296110179/ 296110176/ 296110175/ 296110174/ IMSNU 11154 50145631 680 2838 1779 4354 uid48589 296110178/ 2839 Methanobrevibacter1795593 1818647 148643809/ 148643810/ 148643811/ 148643803/ 148643813/148643814/ smithii ATCC 35061 5015 5632 681 2840 1780 6165 uid58827148643804/ 2841 148643805/ 2842 148643806/ 2843 148643807/ 2844148643808/ 2845 Methanoculleus 985372 997463 397780113/ 397780112/397780111/ 397780110/ 397780109/ 397780108/ bourgensis MS2 5016 5633 6822846 1781 4355 uid171377 Methanolobus 146902 163611 missing 410669356/410669357/ 410669358/ 410669363/ 410669364/ psychrophilus R15 5634 6832847 1782 4356 uid177925 410669360/ 2848 Methanomethylovorans 13591341396372 missing 435851551/ 435851550/ 435851525/ 435851539/ 435851538/hollandica DSM 15978 5635 684 2849 1783 4357 uid184864 435851546/ 2850435851549/ 2851 Methanosarcina 2919153 2939722 missing 20091206/20091205/ 20091204/ 20091198/ 20091197/ acetivorans C2A 5636 685 28521784 4358 uid57879 Methanosarcina mazei 193892 214864 missing 21226255/21226256/ 21226258/ 21226265/ 21226266/ Go1 uid57893 5637 686 2853 17854359 Methanospirillum 1213815 1228530 88602375/ 88602374/ 88602373/88602371/ 88602368/ 88602367/ hungatei JF 1 uid58181 5017 5638 687 28541786 4360 Methanospirillum 2137148 2153998 missing 88603149/ 88603150/88603151/ 88603159/ 88603160/ hungatei JF 1 uid58181 5639 688 2855 17874361 Microlunatus 4106044 4121700 336119446/ 336119447/ 336119448/336119449/ 336139454/ 336119455/ phosphovorus NM 1 5018 5640 689 28561788 4362 uid68055 336119450/ 2857 336119452/ 2858 Moorellathermoacetica 2331989 2347051 83591065/ 83591064/ 83591063/ 83591059/83591058/ 83591057/ ATCC 39073 uid58051 5019 5641 690 2859 1789 436383591062/ 2860 Parvularcula 1068558 1081391 304320704/ 304320703/304320702/ 304320701/ 304320700/ 304320699/ bermudensis 5020 5642 6912861 1790 4364 HTCC2503 uid51641 Pectobacterium 3681934 3716221403059706/ 403059707/ 403059708/ 403059688/ 403059710/ 403059711/carotovorum PCC21 5021 5643 692 2862 1791 4365 uid174335 403059709/ 2863Pelobacter propionicus 2459983 2475529 118580706/ 118580705/ 118580704/118580703/ 118580699/ 118580698/ DSM 2379 uid58255 5022 5644 693 28641792 4366 Pelodictyon 2122302 2136560 194337066/ 194337067/ 194337068/194337069/ 194337071/ 194337072/ phaeoclathratiforme BU 5023 5645 6942865 1793 4367 1 uid58173 Photorhabdus 470261 481491 253987910/253987911/ 253987912/ 253987913/ 253987914/ Missing asymbiotica uid592435024 5646 695 2866 1794 Polaromonas JS666 120587 142418 91790841/91790842/ 91790843/ 91790846/ 91790850/ 91790851/ uid58207 5025 5647 6962867 1795 4368 Pseudomonas 940354 955546 330807432/ 330807433/330807434/ 330807435/ 330807437/ 330807438/ brassicacearum 5026 5648 6972868 1796 4369 NFM421 uid66303 Pseudomonas stutzeri 1382383 1400530392420331/ 392420330/ 392420329/ 392420323/ 392420322/ 392420321/ CCUG29243 5027 5649 698 2869 1797 4370 uid168379 392420328/ 2870Psychrobacter 1973411 1984296 93006435/ 93006434/ 93006433/ 93006432/93006431/ Missing cryohalolentis K5 5028 5650 699 2871 1798 uid58373Rhodococcus 3099107 3111990 226306385/ 226306384/ 226306383/ 226306382/226306381/ 226306380/ erythropolis PR4 5029 5651 700 2872 1799 4371uid59019 Rhodopseudomonas 1186657 1200150 192289534/ 192289535/192289536/ 192289537/ 192289538/ 192289539/ palustris TIE 1 5030 5652701 2873 1800 4372 uid58995 Saccharophagus 3482579 3496946 90022404/90022403/ 90022402/ 90022401/ 90022398/ 90022397/ degradans 2 40 50315653 702 2874 1801 4373 uid57921 Salmonella enterica 4749510 4764672378453463/ 378453462/ 378453461/ 378453460/ 378453457/ 378453456/serovar Typhimurium 5032 5654 703 2875 1802 4374 14028S uid86059Salmonella enterica 4755689 4770857 383499057/ 383499056/ 383499055/383499054/ 383499051/ 383499050/ serovar Typhimurium 5033 5655 704 28761803 4375 798 uid158047 Salmonella enterica 4736677 4751839 16767742/16767741/ 16767740/ 16767739/ 16767736/ 16767735/ serovar Typhimurium5034 5656 705 2877 1804 4376 LT2 uid57799 Salmonella enterica 47572604772422 378702331/ 378702330/ 378702329/ 378702328/ 378702325/378702324/ serovar Typhimurium 5035 5657 706 2878 1805 4377 SL1344uid86645 Salmonella enterica 4757261 4772429 379703735/ 379703734/379703733/ 379703732/ 379703729/ 379703728/ serovar Typhimurium 50365658 707 2879 1806 4378 ST4 74 uid84393 Salmonella enterica 48340594849227 378987165/ 378987364/ 378987163/ 378987162/ 378987159/378987158/ serovar Typhimurium 5037 5659 708 2880 1807 4379 T000240uid84397 Salmonella enterica 4758667 4773829 378447805/ 378447804/378447803/ 378447802/ 378447799/ 378447798/ serovar Typhimurium 50385660 709 2881 1808 4380 uid86061 Salmonella enterica 4697115 4712277378991759/ 378991758/ 378991757/ 378991756/ 378991753/ 378991752/serovar Typhimurium 5039 5661 710 2882 1809 4381 UK 1 uid87049Selenomonas sputigena 820667 833330 330838573/ 330838574/ 330838575/330838576/ 330838578/ Missing ATCC 35185 uid55329 5040 5662 711 28831810 Shewanella ANA 3 2142947 2155062 117920263/ 117920264/ 117920265/117920266/ 117920268/ Missing uid58347 5041 5663 712 2884 1811Shewanella MR 4 2018034 2030135 113970045/ 113970046/ 113970047/113970048/ 113970050/ Missing uid58345 5042 5664 713 2885 1812 Slackia349608 366195 257063043/ 257063042/ 257063041/ 257063038/ 257063040/257063036/ heliotrinireducens DSM 5043 5665 714 2886 1813 4382 20476uid59051 257063039/ 2887 Spirosoma linguale 5893753 5908214 284039689/284039690/ 284039691/ 284039694/ 284039695/ 284039696/ DSM 74 uid434135044 5666 715 2888 1814 4383 Sulfuricurvum kujiense 1691724 1708885313682809/ 313682810/ 313682811/ 313682812/ 313682819/ 313682820/ DSM16994 uid60789 5045 5667 716 2889 1815 4384 313682817/ 2890Synechococcus PCC 875746 901348 427712028/ 427712029/ 427712030/427712018/ 427712035/ 427712036/ 6312 uid182934 5046 5668 717 2891 18164385 427712032/ 2892 Syntrophomonas wolfei 1119627 1137082 114566519/114566520/ 114566521/ 114566522/ 114566529/ 114566530/ Goettingenuid58179 5047 5669 718 2893 1817 4386 114566525/ 2894 114566528/ 2895Syntrophomonas wolfei 2823944 2853705 114568002/ 114568001/ 114568000/114567997/ 114567995/ 114567994/ Goettingen uid58179 5048 5670 719 28961818 4387 114567999/ #N/A 2897 114568012/ #N/A 2898 Syntrophus 932420951906 85858754/ 85858752/ 85858751/ 85858747/ 85858744/ 85858743/aciditrophicus SB 5049 5671 720 2899 1819 4388 uid58539Tepidanaerobacter 565314 579879 438001500/ 438001501/ 438001502/438001506/ 438001508/ 438001509/ acetatoxydans Re1 5050 5672 721 29001820 4389 uid184827 438001507/ 2901 Tepidanaerobacter Re1 564518 579375332798527/ 332798528/ 332798529/ 332798531/ 332798532/ 332798533/uid66873 5051 5673 722 2902 1821 4390 Thauera MZ1T 325210 347552217968744/ 217968745/ 217968746/ 217968749/ 217968754/ 217968755/uid58987 5052 5674 723 2903 1822 4391 Thermacetogenium 2228448 2241200410668523/ 410668522/ 410668521/ 410668520/ 410668519/ 410668518/ phaeumDSM 12270 5053 5675 724 2904 1823 4392 uid177811 Thermoanaerobacterium483298 496321 304316004/ 304316005/ 304316006 304316007/ 304316008/304316009/ thermosaccharolyticum 5054 5676 /725 2905 1824 4393 DSM 571uid51639 Thiocystis violascens 1605822 1624356 390949850/ 390949849/390949848/ 390949845/ 390949842/ Missing DSM 198 uid74025 5055 5677 7262906 1825 Thioflavicoccus mobilis 70557 88066 431933024/ 431933023/431933022/ 431933018/ 431933015/ 431933014/ 8321 uid184343 5056 5678 7272907 1826 4394 431933021/ 2908 Vibrio cholerae MJ 3021293 3036340229608800/ 229608799/ 229608798/ 229608796/ 229608795/ 229608794/ 1236uid59387 5057 5679 728 2909 1827 4395 Vibrio cholerae O1 110486 123671360034501/ 360034502/ 360034503/ 360034504/ 360034505/ 360034506/ 2010EL1786 uid78933 5058 5680 729 2910 1828 4396 Zymomonas mobilis 15901551602973 260753603/ 260753604/ 260753605/ 260753606/ 260753607/260753608/ NCIMB 11163 5059 5681 730 2911 1829 4397 uid41019Magnetospirillum 2215526 2231935 83311137/ 83311138/ 83311139/ 83311143/83311145/ 83311146/ magneticum AMB 1 5060 5682 731 2912 1830 4398uid58527 83311142/ 732 Marinobacter aquaeolei 627682 647744 120553492/120553493/ 120553494/ 120553498/ 120553501/ 120553502/ VT8 uid59419 50615683 733 2913 1831 4399 Nostoc punctiforme 6986741 7000722 186685714/186685707/ 186685708/ 186685709/ 186685712/ 186685713/ PCC 73102uid57767 5062 5684 734 2914 1832 4400 Rhodobacter 628475 643049146279749/ 146279748/ Rsph17025_3734/ 146279745/ 146279744/ 146279743/sphaeroides ATCC 5063 5685 615 2915 1833 4401 17025 uid58451 Runellaslithyformis 4922398 4938169 338213691/ 338213690/ 338213689/ 338213688/338213683/ 338213682/ DSM 19594 uid68317 5064 5686 735 2916 1834 4402Acidovorax sp. NO-1 4749 20769 365096841/ 365096842/ 365096843/365096846/ 365096849/ 365096850/ 5336 5905 1072 3214 2158 4265Acinetobacter 33402 52203 421624927/ 421624915/ 421624973/ 421624892/421624935/ 421624974/ baumannii OIFC098 5341 5911 1078 3219 2164 4271Acinetobacter 57480 72041 427425605/ 427425618/ 427425586/ 427425611/427425576/ 427425548/ baumannii WC-136 5342 5912 1079 3220 2165 4272Acinetobacter sp. P8-3- 1850 16994 358012810/ 358012809/ 358012808/358012807/ 358012805/ 358012804/ 8 5326 5895 1062 3204 2147 4256Actinomyces neuii 83071 96264 405982588/ 405982587/ 405982586/405982584/ 405982583/ 405982582/ BVS029A5 5356 5927 1094 3235 2180 4287405982585/ 3236 Aurantimonas 1067851 1080860 90418999/ 90418998/90418997/ 90418996/ 90418995/ 90418994/ manganoxydans SI85- 5319 58881055 3197 2140 4250 9A1 Bacillus cereus 89926 103011 206975566/206975742/ 206975617/ 206975739/ 206975748/ 206975706/ H3081.97 53065875 1041 3181 2126 4236 Bacteroides ovatus SD 22479 38083 294645431/294645432/ 294645433/ 294645435/ 294645438/ 294645439/ CC 2a 5364 59361103 3246 2189 4295 294645434/ 1104 Bacteroides sp. 2_1_7 444155 466254255014563/ 255014564/ 255014565/ 255014566/ 255014573/ 255014574/ 53125881 1048 3190 2133 4243 Bacteroides sp. 15816 30563 265757021/265757020/ 265757019/ 265757018/ 265757016/ 265757015/ 3_1_33FAA 53295898 1065 3207 2150 4259 Bacteroides sp. D2 958538 975774 383114785/383114786/ 383114787/ 383114788/ 383114792/ 383114793/ 5327 5896 10633205 2148 4257 Bacteroides sp. 2_1_22 149553 165156 262405135/262405136/ 262405137/ 262405138/ 262405141/ 262405142/ 5346 5916 10833224 2169 4276 Clostridium perfringens 27368 50007 169343970/ 169343964/169343946/ 169343972/ 169343967/ 169343977/ C str. JGS1495 5355 59261093 3234 2179 4286 Collinsella aerofaciens 42484 56961 139439063/139439064/ 139439065/ 139439066/ 139439069/ 139439070/ ATCC 25986 53095878 1045 3186 2130 4240 139439068/ 3187 Coprobacillus sp. 950856 973460365831804/ 365831805/ 365831806/ 365831813/ 365831816/ 365831817/3_3_56FAA 5367 5939 1109 3251 2192 4298 365831811/ 1110Desulfonatronospira 1059989 1073987 missing 298528557/ 298528556/298528555/ 298528552/ 298528551/ thiodismutans ASO3-1 5906 1073 32152159 4266 Enterobacter 1757377 1772326 334125669/ 334125670/ 334125671/334125672/ 334125675/ 334125676/ hormaechei ATCC 5289 5859 1023 31592108 4218 49162 Enterococcus faecium 192 13547 425040991/ 425040992/425040993/ 425040994/ 425040995/ 425040996/ 511 5316 5885 1052 3194 21374247 Enterococcus faecium 141305 154660 431534711/ 431534712/ 431534713/431534714/ 431534715/ 431534716/ E1731 5294 5864 1028 3164 2113 4223Enterococcus faecium 294802 308157 431753899/ 431753900/ 431753901/431753902/ 431753903/ 431753904/ E2883 5357 5928 1095 3237 2181 4288Enterococcus faecium 192 13547 314941479/ 314941480/ 314941481/314941482/ 314941483/ 314941484/ TX0133C 5310 5879 1046 3188 2131 4241Escherichia coli 4.0522 178149 191595 417191592/ 417191528/ 417191550/417191518/ 417191624/ 417191569/ 5344 5914 1081 3222 2167 4274Escherichia coli B41 177464 190841 417293178/ 417293264/ 417292957/417293087/ 417293167/ 417293248/ 5358 5929 1096 3238 2182 4289Escherichia coli B799 163075 176512 423709631/ 423709630/ 423709629/423709628/ 423709627/ 423709626/ 5291 5861 1025 3161 2110 4220Escherichia coli 68462 81854 419343549/ 419343548/ 419343546/ 419343544/419343543/ 419343542/ DEC13A 5366 5938 1107 3249 2191 4297 419343547/419343545/ 1108 3250 Escherichia coli 154888 168171 419368024/419368147/ 419368080/ 419368110/ 419368013/ 419368125/ DEC13E 5365 59371105 3247 2190 4296 419368151/ 419368121/ 1106 3248 Escherichia coli 50213913 419383946/ 419383945/ 419383944/ 419383942/ 419383941/ 419383940/DEC14C 5299 5869 1033 3171 2118 4228 419383943/ 3172 Escherichia coli165882 179328 419230123/ 419230122/ 419230121/ 419230120/ 419230119/419230118/ DEC9A 5347 5918 1085 3226 2171 4278 Escherichia coli 126845140290 419240873/ 419240872/ 419240871/ 419240869/ 419240868/ 419240867/DEC9C 5298 5868 1032 3169 2117 4227 419240870/ 3170 Escherichia coli302405 315851 419246584/ 419246583/ 419246582/ 419246581/ 419246580/419246579/ DEC9D 5339 5909 1076 3217 2162 4269 Escherichia coli 5053363979 419894759/ 419894758/ 419894757/ 419894756/ 419894755/ 419894754/O111:H8 str. CVM9574 5350 5921 1088 3229 2174 4281 Escherichia coli70169 83615 420088377/ 420088376/ 420088375/ 420088374/ 420088373/420088372/ O111:H8 str. CVM9602 5303 5873 1038 3178 2123 4233Faecalibacterium 28608 45887 160944222/ 160944223/ 160944224/ 160944228/160944230/ 160944231/ prausnitzii M21/2 5325 5894 1061 3203 2146 4255Fusobacterium 64292 79742 419841769/ 419841708/ 419843735/ 419841801/419841754/ Missing necrophorum subsp 5361 5932 1099 3241 2185Fusobacterium sp. 435830 468937 340752713/ 340752714/ 340752704/340752707/ 340752711/ 340752712/ 2_1_31 5317 5886 1053 3195 2138 4248Fusobacterium sp. 7_1 589947 618478 237744585/ 237744584/ 237744599/237744596/ 237744587/ 237744586/ 5354 5925 1092 3233 2178 4285Glaciecola lipolytica E3 6001 19207 410637162/ 410637163/ 410637164/410637165/ 410637166/ 410637167/ 5295 5865 1029 3165 2114 4224Lachnospiraceae 81164 93860 373469122/ 373469123/ 373469124/ 373469126/373469127/ Missing bacterium oral taxon 5332 5901 1068 3210 2153 082 str373469128/ 2154 Lactobacillus casei 4097 20782 418008633/ 418008632/418008631/ 418008628/ 418008627/ 418008626/ UW4 5293 5863 1027 3162 21124222 418008630/ 3163 Lactobacillus helveticus 82009 98806 260101570/260101569/ 260101568/ 260101567/ 260101564/ Missing DSM 20075 5323 58921059 3201 2144 Lactobacillus zeae 53700 70162 366087504/ 366087503/366087502/ 366087499/ 366087498/ 366087497/ KCTC 3804 5363 5935 11023244 2188 4294 366087501/ 3245 Magnetospirillum sp. 2351 15314458920178/ 458920177/ 458920176/ missing 458920175/ 458920174/ SO-1 53385908 1075 2161 4268 Magnetospirillum sp. 12059 25247 458913831/458913830/ 458913829/ 458913827/ 458913826/ 458913825/ SO-1 5343 59131080 3221 2166 4273 Marinobacter sp. 23845 43291 126665997/ 126665996/126665995/ 126665993/ 126665990/ 126665989/ ELB17 5290 5860 1024 31602109 4219 Methanoplanu 1594401 1624870 missing 374629430/ 374629429/374629424/ 374629423/ 374629422/ 5870 1035 3174 2120 4230 374629428/3175 Methanoplanu 2294064 2309549 374630020/ 374630019/ 374630018/374630017/ 374630014/ 374630013/ 5301 5871 1036 3176 2121 4231Methylophaga 75838 91891 335043949/ 335043948/ 335043947/ 335043946/335043943/ 335043942/ aminisulfidivorans MP 5349 5920 1087 3228 21734280 Oribacterium sp. ACB7 598004 613860 363897445/ 363897444/363897443/ 363897440/ 363897438/ 363897437/ 5296 5866 1030 3166 21154225 363897442/ 3167 Oribacterium sp. ACB8 361812 375959 395208378/395208454/ 395208610/ 395208247/ 395208500/ 395208399/ 5337 5907 10743216 2160 4267 Photobacterium sp. 74472 92346 89073849/ 89073848/89073847/ 89073841/ 89073839/ 89073838/ SKA34 5352 5923 1090 3231 21764283 Proteus mirabilis 764883 778479 425069874/ 425069875/ 425069876/425069878/ 425069879/ 425069880/ WGLW6 5328 5897 1064 3206 2149 4258Pseudomonas fragi A22 86067 103151 402700379/ 402700380/ 402700381/402700382/ 402700385/ 402700386/ 5322 5891 1058 3200 2143 4253Pseudomonas 2117 19238 404399334/ 404399333/ 404399332/ 404399331/404399328/ 404399327/ fuscovaginae UPB0736 5331 5900 1067 3209 2152 4261Roseburia inulinivorans 2013 15569 225374528/ 225374527/ 225374526/225374525/ 225374523/ 225374522/ DSM 16841 5334 5903 1070 3212 2156 4263Salmonella enterica 1280 14693 437842753/ 437842752/ 437842751/ missing437842750/ 437842749/ subsp. enterica serovar 5292 5862 1026 2111 4221Enteritidi Salmonella enterica 39286 52691 167551248/ 167551249/167551250/ 167551251/ 205357107/ 167551253/ subsp. enterica serovar 53355904 1071 3213 2157 4264 Saintpau Salmonella enterica 28163 43331427646246/ 427646245/ 427646244/ 427646243/ 427646240/ 427646239/ subsp.enterica serovar 5330 5899 1066 3208 2151 4260 Typhimuriu Salmonellaenterica 72 15632 417522348/ 417522347/ 417522346/ 417522343/ 417522342/417522341/ subsp. enterica serovar 5307 5877 1043 3183 2128 4238 Ugandastr 417522344/ 3184 Sinorhizobium meliloti 157136 171638 418398872/418398871/ 418398870/ 418398867/ 418398866/ 418398865/ CCNWSX0020 53205889 1056 3198 2141 4251 Sphingobium 37182 50024 381199745/ 381199746/381199747/ 381199748/ 381199749/ 381199750/ yanoikuyae XLDN2-5 5321 58901057 3199 2142 4252 Vibrio cholera CIRS 6477 19662 255743726/ missing255743727/ 255743728/ 255743729/ 255743730/ 101 5304 1039 3179 2124 4234Vibrio cholerae 131961 147079 422306078/ 422306079/ 422306080/422306082/ 422306083/ 422306084/ CP1035(8) 5313 5882 1049 3191 2134 4244Vibrio cholerae HC- 4662 17826 423152696/ 423152697/ 423152698/423152699/ 423152700/ 423152701/ 22A1 5360 5931 1098 3240 2184 4291Vibrio cholerae HC- 7323 20508 418336064/ 418336065/ 418336066/418336067/ 418336068/ 418336069/ 23A1 5359 5930 1097 3239 2183 4290Vibrio cholerae HC- 4645 17830 418342900/ 418342901/ 418342902/438342903/ 418342904/ 418342905/ 28A1 5302 5872 1037 3177 2122 4232Vibrio cholerae HC- 4645 17830 423155502/ 423155503/ 423155504/423155505/ 423155506/ 423155507/ 32A1 5340 5910 1077 3218 2163 4270Vibrio cholerae HC- 594 12995 missing 422890702/ 422890703/ 422890704/422890705/ 422890706/ 40A1 5917 1084 3225 2170 4277 Vibrio cholerae HC-4639 17824 424621185/ 424621186/ 424621187/ 424621188/ 424621189/424621190/ 47A1 5345 5915 1082 3223 2168 4275 Vibrio cholerae HC- 54712948 missing 423164023/ 423164024/ 423164025/ 423164026/ 423164027/48B2 5876 1042 3182 2127 4237 Vibrio cholerae HC- 56848 70060 424015508/424015509/ 424015510/ 424015511/ 424015512/ 424015513/ 55B2 5348 59191086 3227 2172 4279 Vibrio cholerae HC- 56864 70076 423878984/423878985/ 423878986/ 423878987/ 423878988/ 423878989/ 60A1 5318 58871054 3196 2139 4249 Vibrio cholerae HC- 57077 70289 419832015/ missing419832016/ 419832017/ 419832018/ 419832019/ 61A2 5308 1044 3185 21294239 Vibrio cholerae HC- 4639 17824 443514284/ 443514285/ 443514286/443514287/ 443514288/ 443514289/ 68A1 5324 5893 1060 3202 2145 4254Vibrio cholerae HC- 4637 17822 443518098/ 443518099/ 443518100/443518101/ 443518102/ 443518103/ 71A1 5305 5874 1040 3180 2125 4235Vibrio cholerae HC- 4635 17820 443534356/ 443534357/ 443534358/443534359/ 443534360/ 443534361/ 80A1 5333 5902 1069 3211 2155 4262Vibrio cholerae HC- 4631 17816 424655756/ 424655757/ 424655758/424655759/ 424655760/ 424655761/ 81A2 5288 5858 1022 3158 2107 4217Vibrio cholerae 7324 20488 417815509/ 417815510/ 417815511/ 417815512/417815513/ 417815514/ HCUF01 5315 5884 1051 3193 2136 4246 Vibriocholerae MO10 324004 339140 254850926/ 254850927/ 254850928/ 254850931/254850932/ 254850933/ 5314 5883 1050 3192 2135 4245 Vibrio cholerae O1str. 4654 17839 472152150/ 472152151/ 472152152/ 472152153/ 472152154/472152155/ EC-0012 5297 5867 1031 3168 2116 4226 Vibrio cholerae O1 str.4648 17833 472383764/ 472183765/ 472183766/ 472183767/ 472183768/472183769/ EM-1546 5287 5857 1021 3157 2106 4216 Vibrio cholerae O1 str.4648 17833 472202869/ 472202870/ 472202871/ 472202872/ 472202873/472202874/ NHCC-006C 5351 5922 1089 3230 2175 4282 Vibrio cholerae HC-44947 58111 421338424/ 421338423/ 421338422/ 421338421/ 421338420/421338419/ 20A2 5362 5933 1100 3242 2186 4292 Vibrio cholerae HC- 4226755452 423147143/ 423147142/ 423147141/ 423147140/ 423147139/ 423147138/21A1 5311 5880 1047 3189 2132 4242 Vibrio cholerae HC- 42245 55430424618577/ 424618576/ 424618575/ 424618574/ 424618573/ 424618572/ 42A15353 5924 1091 3232 2177 4284 Vibrio cholerae HC- 58485 71697 424630791/missing 424630790/ 424630789/ 424630788/ 424630787/ 51A1 5300 1034 31732119 4229 Xanthomonas 3685 17230 missing 325918008/ 325918007/325918006/ 325918004/ 325918003/ vesicatoria ATCC 5934 1101 3243 21874293 35937 Acetivibrio 169996 184667 366164462/ 366164461/ 366164460/366164459/ 366164456/ 366164455/ cellulolyticus CD2 5174 5766 893 30311982 4105 Acinetobacter 10653 23830 457996552/ 457996553/ 457996554/457996555/ 457996556/ 457996557/ baumannii AB5256 5162 5755 880 30181969 4092 Acinetobacter 55392 70524 417550475/ 417550474/ 417550828/417550745/ 417551043/ 417550931/ baumannii Naval-18 5190 5780 910 30491999 4120 Acinetobacter johnsonii 37452 52595 262371249/ 262371248/262371247/ 262371246/ 262371244/ 262371243/ SH046 5119 5720 836 29741925 4052 Acinetobacter junii 131814 145059 262374463/ 262374464/262374465/ 262374466/ 262374467/ 262374468/ SH205 5121 5722 838 29761927 4053 Acinetobacter lwoffii 39119 53440 262377711/ 262377712/262377713/ 262377714/ 262377716/ 262377717/ SH145 5181 5772 901 30391990 4031 262377715/ 3040 Acinetobacter 6083 19313 421857671/ 421857672/421857673/ 421857674/ 421857675/ 421857676/ radioresistens DSM 5141 5738859 2996 1948 4073 6976 = NBRC 102413 Acinetobacter sp. SH024 1005924620 293611304/ 293611305/ 293611306/ 293611307/ 293611309/ 293611310/5184 5775 904 3043 1993 4114 Actinomyces sp. ICM47 13893 28963396584771/ 396584768/ 396584764/ 396584767/ 396584777/ 396584762/ 51025703 818 2954 6204 4036 Actinomyces sp. oral 1582915 1598023 320095079/320095080/ 320095081/ 320095082/ 320095085/ 320095086/ taxon 178 str5139 5735 857 2994 1946 4072 Alcanivorax pacificus 73364 89677407801754/ 407801755/ 407801756/ 407801760/ 407801761/ 407801762/ W11-55128 5727 845 2982 1934 4060 Alcanivorax sp. DG881 3121028 3136375254429639/ 254427953/ 254428479/ 254429762/ 254428445/ 254426952/ 51655758 883 3022 1972 4095 Bacillus cereus 790081 800966 423480947/423480948/ 423480949/ 423480950/ 423480951/ Missing BAG6X1-2 5252 5831976 3116 2065 Bacillus cereus HuB2-9 3250049 3263169 423535446/423535445/ 423535444/ 423535443/ 423535442/ 423535441/ 5246 5826 9693108 2058 4173 Bacteroides coprophilus 12964 26509 224023324/ 224023323/224023322/ 224023320/ 224023319/ 224023318/ DSM 18228 5281 5700 10096194 2098 4209 Bacteroides ovatus 2284234 2301803 423288076/ 423288075/423288074/ 423288073/ 423288070/ 423288069/ CL02T12C04 5150 5744 8683005 1957 4081 Bacteroides ovatus SD 59559 81656 293373691/ 293373690/293373689/ 293373688/ 293373679/ 293373678/ CMC 3f 5144 5740 862 29991951 4076 Bacteroides sp. 1_1_14 157533 186706 298384296/ 298384295/298384294/ missing 298384292/ 298384291/ 5282 5851 1011 2099 4210Bacteroides sp. D1 413277 428835 345508162/ 237715294/ 345508160/237715296/ 345508156/ 345508155/ 5278 5699 1006 6196 2095 4207Bacteroides 90276 105878 294807073/ 294807072/ 294807071/ 294807069/294807066/ 294807065/ xylanisolvens SD CC 5276 5698 1003 6190 2092 42041b Bifidobacterium 878442 897214 229817499/ 229817500/ 229817501/ZP_03449450/ 229817507/ 229817508/ angulatum DSM 20098 5126 5726 8436192 1932 4058 Bifidobacterium 7024 23499 421735410/ 421735409/421735408/ 421735407/ 421735404/ 421735403/ bifidum IPL 5143 5739 8612998 1950 4075 Bifidobacterium 939991 958805 313140117/ 313140118/313140119/ 313140120/ 313140123/ 313140124/ bifidum NCIMB 41171 51695760 887 3026 1976 4099 Brachybacterium 53546 66327 386775206/386775207/ 386773208/ 386775209/ 386775210/ 386775211/ paraconglomeratum5214 5799 936 3076 2025 4143 LC44 Brevibacterium 42197 55309 295396113/295396312/ 295396111/ 295396110/ 295396108/ 295396107/ mcbrellneri ATCC5199 5789 920 3060 2009 4129 49030 Cellvibrio sp. BR 223559 242477388258095/ 388258094/ 388258093/ 388258089/ 388258086/ 388258085/ 51915781 911 3050 2000 4121 388258091/ 3051 Clostridium butyricum 11585711173561 237668144/ 237668630/ 237667881/ 237668455/ 237668352/237667216/ E4 str. BoNT E BL5262 5189 5779 909 3048 1998 4119Clostridium perfringens 53370 80361 182624257/ 182624189/ 182624228/182624175/ 182624223/ 182624227/ D str. JGS1721 5218 5803 940 3080 20294146 Collinsella intestinalis 30782 46221 229814947/ 229814946/229814945/ 229814944/ 229814938/ 229814937/ DSM 13280 5157 5750 875 30131964 4087 Collinsella stercoris 31011 46881 210635259/ 210635260/210635261/ 210635262/ 210635282/ 210635283/ DSM 13279 5235 5818 958 30972047 4163 Coprobacillus sp. 100734 114652 374625977/ 374625978/374625979/ 374625981/ 374625983/ 374625984/ 8_2_54BFAA 5107 5708 8232959 1911 4040 Cylindrospermopsis 2528 15922 missing 282900779/282900778/ 282900774/ 282900772/ 282900771/ raciborskii CS-505 5855 10196198 2104 4215 delta proteobacterium 35975 51793 301064129/ truncated301064167/ 301064133/ 301064130/ 301064155/ NaphS2 5195 915 3055 20044125 Desulfotomaculum 100510 112411 357040623/ 357040624/ 357040625/357040626/ 357040629/ Missing gibsoniae DS 5211 5796 932 3072 2021Enterobacter sp. SST3 61939 75308 401676813/ 401676814/ 401676815/401676816/ 401676817/ 401676818/ 5243 5824 966 3105 2055 4170Enterococcus faecalis 21896 38028 307287298/ 307287299/ 307287300/307287301/ 307287305/ 307287306/ TX0109 5132 5730 850 2987 1939 4065Enterococcus faecalis 21900 38032 422702471/ 422702472/ 422702473/422702474/ 422702478/ 422702479/ TX1302 5237 5820 960 3099 2049 4164Enterococcus faecium 9609 22964 425047892/ 425047891/ 425047890/425047889/ 425047888/ 425047887/ 509 5407 5797 934 3074 2023 4141Enterococcus faecium 192 13547 425034027/ 425034028/ 425034029/425034030/ 425034031/ 425034032/ 514 5164 5757 882 3021 1971 4094Enterococcus faecium 5282 18637 425024182/ 425024181/ 425024180/425024179/ 425024178/ 425024177/ C1904 5405 5785 916 3056 2005 4126Enterococcus faecium 142 13497 425019537/ 425019538/ 425019539/425019540/ 425019541/ 425019542/ C497 5151 5745 869 3006 1958 4030Enterococcus faecium 308557 323090 430834457/ 430834456/ 430834455/430834454/ 430834452/ 430834451/ E0679 5187 5777 907 3046 1996 4117Enterococcus faecium 314134 328775 431640912/ 431640911/ 431640910/431640909/ 431639958/ 431639957/ E1904 5265 5841 990 3133 2079 4191Enterococcus faecium 5283 18638 424979863/ 424979862/ 424979861/424979860/ 424979859/ 424979858/ ERV99 5399 5709 824 2960 1912 4041Enterococcus faecium 191 13546 424977969/ 424977970/ 424977971/424977972/ 424977973/ 424977974/ P1123 5203 5792 924 3064 2013 4132Enterococcus faecium 5282 18637 424974922/ 424974921/ 424974920/424974919/ 424974918/ 424974917/ P1137 5414 5848 1001 3144 2090 4202Enterococcus faecium 191 13546 424971162/ 424971163/ 424971164/424971165/ 424971166/ 424971167/ P1139 5204 5793 925 3065 2014 4133Enterococcus faecium 9607 22962 314950597/ 314950596/ 314950595/314950594/ 314950593/ 314950592/ TX0133A 5404 5768 895 3033 1984 4106Enterococcus faecium 192 13547 314996683/ 314996684/ 314996685/314996686/ 314996687/ 314996688/ TX0133a01 5244 5825 967 3106 2056 4171Enterococcus faecium 9609 22964 314939181/ 314939180/ 314939179/314939178/ 314939177/ 314939176/ TX0133a04 5409 5827 970 3109 2059 4174Enterococcus faecium 9609 22964 314992451/ 314992450/ 314992449/314992448/ 314992447/ 314992446/ TX0133B 5408 5804 941 3081 2030 4147Escherichia coli 2534- 70489 83934 417589503/ 417589502/ 417589501/417589499/ 417589498/ 417589497/ 86 5275 5849 1002 3145 2091 4203417589500/ 3146 Escherichia coli 3.3884 1217008 1230445 417270173/417270040/ 417269258/ 417268711/ 417269719/ 417270270/ 5116 5717 8332970 1922 4049 Escherichia coli 96.154 246141 259589 417223908/417224699/ 417224010/ 417225276/ 417224519/ 417225329/ 5285 5854 10163155 2102 4213 417225258/ 1017 Escherichia coli 318305 331717 419281130/419281129/ 419281128 419281126/ 419281125/ 419281124/ DEC10E 5118 57192972 1924 4051 419281127/ 2973 Escherichia coli 147401 160847 419281503/419281502/ 419281501 419281500/ 419281499/ 419281498/ DEC10F 5225 58093088 2037 4154 Escherichia coli 92227 105618 419352610/ 419352609/419352607/ 419352604/ 419352603/ 419352602/ DEC13B 5284 5853 1014 31532101 4212 419352608/ 419352606/ 1015 3154 Escherichia coli 68089 81481419358139/ 419358138/ 419358136/ 419358134/ 419358133/ 419358132/ DEC13C5283 5852 1012 3151 2100 4211 419358137/ 419358135/ 1013 3152Escherichia coli 138809 154330 419363104/ 419363100/ 419363098/419363096/ 419363095/ 419363094/ DEC13D 5277 5850 1004 3147 2093 4205419363099/ 419363097/ 1005 3148 Escherichia coli 143274 156684419378584/ 419378583/ 419378582/ 419378580/ 419378578/ 419378576/ DEC14B5250 5829 974 3113 2063 4178 419378581/ 3114 Escherichia coli 9353 22765419389188/ 419389187/ 419389186/ 419389184/ 419389183/ 419389182/ DEC14D5156 5749 874 3011 1963 4086 419389185/ 3012 Escherichia coli 1132824717 419173689/ 419173698/ 419173682/ 419173671/ 419173696/ 419173675/DEC7B 5109 5710 826 2962 1914 4043 Escherichia coli 78187 91632419200151/ 419200150/ 419200149/ 419200148/ 419200147/ 419200146/ DEC8A5200 5790 921 3061 2010 4130 Escherichia coli 18788 33547 419206519/419206636/ 419206641/ 419206658/ 419206669/ 419206621/ DEC8B 5227 5811950 3090 2039 4156 419206666/ 5812 Escherichia coli 139979 153424419235451/ 419235547/ 419235503/ 419235607/ 419235606/ 419235530/ DEC9B5124 5724 841 2979 1930 4056 419235615/ 2980 Escherichia coli DEC9E26957 40402 419252319/ 419252318/ 419252317/ 419252315/ 419252314/419252313/ 5261 5838 985 3127 2074 4187 419252316/ 3128 Escherichia coliJB1-95 70264 85023 417209283/ 417209341/ 417209350/ 417209346/417209331/ 417209313/ 5267 5842 992 3135 2081 4193 Escherichia coli KD214059 28959 419919262/ 419919261/ 419919260/ 419919259/ 419919256/419919255/ 5403 5761 888 3027 1977 4100 Escherichia coli KTE12 333731347168 432379522/ 432379521/ 432379520/ 432379519/ 432379518/ 432379517/5106 5707 822 2958 1910 4039 Escherichia coli 791 14168 433099196/433099195/ 433099194/ 433099193/ 433099192/ 433099191/ KTE139 5154 5748872 3009 1961 4084 Escherichia coli 3821 17182 433318462/ 433118461/433118460/ 433118459/ 433118458/ 433118457/ KTE153 5401 5729 849 29861938 4064 Escherichia coli 421523 434900 432983125/ 432983126/432983127/ 432983128/ 432983129/ 432983130/ KTE211 5161 5754 879 30171968 4091 Escherichia coli 593208 606594 432993615/ 432993616/432993617/ 432993618/ 432993619/ 432993620/ KTE218 5198 5788 919 30592008 4128 Escherichia coli 445622 459002 432531967/ 432531966/432531965/ 432531964/ 432531963/ 432531962/ KTE234 5239 5822 962 31012051 4166 Escherichia coli 290106 305021 432551900/ 432551901/432551902/ 432551903/ 432551906/ 432551907/ KTE47 5233 5816 956 30952045 4161 Escherichia coli 325634 339020 432566734/ 432566735/432566736/ 432566737/ 432566738/ 432566739/ KTE53 5264 5840 989 31322078 4190 Escherichia coli 789918 803295 432708021/ 432708022/432708023/ 432708024/ 432708025/ 432708026/ KTE6 5183 5774 903 3042 19924113 Escherichia coli MS 69- 8280 23192 301024309/ 301024308/ 301024307/301024306/ 301024303/ 301024302/ 1 5147 5742 865 3002 1954 4079Escherichia coli 1722 15111 442594978/ 442594979/ 442594980/ 442594981/442594982/ 442594983/ O10:K5(L):H4 str. 5159 5752 877 3015 1966 4089ATCC 23506 Escherichia coli 43889 57335 419887469/ 419887468/ 419887467/419887466/ 419887465/ 419887464/ O111:H8 str. CVM9570 5220 5806 943 30832032 4149 Escherichia coli 13485 26931 420093467/ 420093468/ 420093469/420093470/ 420093471/ 420093472/ O111:H8 str. CVM9634 5134 5732 852 29891941 4067 Escherichia coli 111648 125096 458688575/ 458688574/458688573/ 458688572/ 458688571/ 458688570/ O113:H21 str. CL-3 5271 5844996 3139 2085 4197 Escherichia coli 179 13555 458890352/ 458890353/458890354/ 483041219/ 458890355/ 458890356/ O25b:ST131 str. JIE186 51635756 881 3019 1970 4093 481041220/ 3020 Escherichia coli 263790 277236415823690/ 415823689/ 415823688/ 415823687/ 415823686/ 415823685/ OK11805194 5784 914 3054 2003 4124 Escherichia coli S17 4631 19699 450256213/450256210/ 450256209/ 450256207/ 450256206/ 450256205/ 5168 5759 8863025 1975 4098 Escherichia coli 133003 146449 417605265/ 417605264/417605263/ 417605261/ 417605260/ 417605259/ STEC_94C 5113 5714 830 29661919 4046 417605262/ 2967 Escherichia coli 209301 222747 458059912/458059911/ 458059910/ 458059909/ 458059908/ 458059907/ TW10828 5138 5734856 2993 1945 4071 Eubacterium 2986470 3002126 389577853/ 389577852/389577851/ 389577850/ 389577845/ Missing cellulosolvens 6 5216 5801 9383078 2027 Faecalibacterium 463318 476823 257438028/ 257438027/257438026/ 257438025/ 257438023/ 257438022/ prausnitzii A2-165 5192 5782912 3052 2001 4122 Fulvimarina pelagi 98942 111984 114706333/ 114706332/114706331/ 114706330/ 114706329/ 114706328/ HTCC2506 5193 5783 913 30532002 4123 Fusobacterium 564908 579712 373113611/ 373113608/ 373113625/373113618/ 373113613/ Missing necrophorum subsp 5140 5736 858 2995 1947373113609/ 5737 Fusobacterium 33577 58056 296328615/ 296328614/296328627/ 296328621/ 296328617/ 296328616/ nucleatum subsp. 5179 5770899 3037 1988 4110 nucleatum ATCC 23726 Fusobacterium sp. 557323 577812336418217/ 336418218/ 336418208/ 336418211/ 336418215/ 336418216/ 11_3_25188 5778 908 3047 1997 4118 Fusobacterium sp. 1001099 1027629294785575/ 294785574/ 294785588/ 294785585/ 294785577/ 294785576/ 3_1_275180 5771 900 3038 1989 4111 Fusobacterium sp. 242664 257240 317058273/317058274/ 317058268/ 317058270/ 317058272/ Missing 3_1_5R 5175 5767 8943032 1983 Fusobacterium ulcerans 320598 335972 404368581/ 404368582/404368577/ 404368578/ 404368579/ 404368580/ ATCC 49185 5223 5807 9463086 2035 4152 gamma proteobacterium 205372 220880 386288256/ 386288257/386288258/ 386288262/ 386288263/ 386288264/ BDW918 5172 5764 891 30301980 4103 gamma proteobacterium 77590 90394 329896084/ 329896083/329896082/ 329896081/ 329896080/ 329896079/ IMCC3088 5131 5728 848 29851937 4063 Geobacillus sp. 128797 142893 196250359/ 196250358/ 196250357/196250356/ 196250354/ Missing G11MC16 5232 5815 955 3094 2044Geobacillus 1816670 1827544 423719991/ 423719992/ 423719993/ 423719994/423719995/ Missing thermoglucosidan 5201 5791 922 3062 2011 Glaciecolapolaris LMG 41332 52442 410618148/ 196250358/ 410618146/ 410618145/410618144/ Missing 21857 5280 5815 1008 3150 2097 Glaciecola punicea141624 156305 381395614/ 381395615/ 381395616/ 381395619/ 381395620/381395621/ DSM 14233 = ACAM 5236 5819 959 3098 2048 4033 611 Haloarculajaponica 437852 452733 448689859/ 448689858/ 448689857/ 448689855/448689853/ 448689852/ DSM 6131 5254 5833 978 3119 2067 4180 Halomonassp. HAL1 3095 24448 352101114/ 352101113/ 352101112/ 5′ of gene352101104/ 352101103/ 5125 5725 842 missing 1931 4057 Holdemaniafiliformis 5004 17955 223986440/ 223986439/ 223986438/ 223986437/223986436/ 223986435/ DSM 12042 5186 5776 906 3045 1995 4116 Holophagafoetida 8075 23271 373485785/ 373485786/ 373485787/ 373485789/373485791/ 373485792/ DSM 6591 5115 5716 832 2969 1921 4048 Johnsonellaignava 86362 100649 358066716/ 358066715/ 358066714/ 358066713/358066709/ 358066708/ ATCC 51276 5240 5823 963 3102 2052 4167Lachnoanaerobaculum 1084944 1098333 315651036/ 315651037/ 315651038/315651039/ 315651043/ 315651044/ (Eubacterium) 5406 5944 1172 3278 22484024 saburreum DSM 3986 Lachnoanaerobaculum 2724 17869 419720906/419720918/ 419720908/ 419720916/ 419720919/ 419720903/ (Eubacterium)5400 5944 1170 3276 2248 4020 saburreum F0468 Lachnospiraceae 88155102869 336440367/ 336440366/ 336440365/ 336440363/ 336440358/ Missingbacterium 1_1_57FAA 5105 5706 821 2957 1909 Lachnospiraceae 229861244575 317501016/ 317501017/ 317501018/ 317501020/ 317501025/ Missingbacterium 8_1_57FAA 5148 5743 866 3003 1955 Lachnospiraceae oral 111528132974 331001729/ 331001730/ 331001731/ 331001734/ 331001737/ Missingtaxon 107 str 5110 5711 827 2963 1915 331001745/ 1916 Lactobacilluscasei Lpc-37 1267 17585 418013531/ 418013530/ 418013529/ 418013528/418013525/ 418013524/ 5182 5773 902 3041 1991 4112 Lactobacillus 142681159146 421768513/ 421768512/ 421768511/ 421768508/ 421768507/ 421768506/rhamnosus LRHMDP2 5255 5834 979 3120 2068 4181 421768510/ 3121Lactobacillus 129821 146256 421772571/ 421772572/ 421772573/ 421772574/421772577/ 421772578/ rhamnosus LRHMDP3 5253 5832 977 3117 2066 4035421772576/ 3118 Lactobacillus johnsonii 547785 564068 227889974/227889975/ 227889976/ 227889977/ 227889982/ Missing ATCC 33200 5196 5786917 3057 2006 Lactobacillus reuteri 141036 153317 227544819/ 227544818/227544817/ 227544816/ 227544813/ Missing CF48-3A 5411 5839 986 3129 2075Microcystis aeruginosa 76626 89380 missing 425444979/ 425444980/425444983/ 425444986/ Missing PCC 9443 5856 1020 3156 2105Nitratireductor indicus 129343 142197 407974990/ 407974989/ 407974988/407974987/ 407974986/ 407974985/ C115 5160 5753 878 3016 1967 4090Opitutacea 241740 261922 373849354/ missing 373849352/ missing373849343/ 373849342/ 5279 1007 2096 4208 Oribacterium sp. ACB1 648540662687 363899397/ 363899396/ 363899395/ 363899394/ 363899392/ 363899391/5117 5718 834 2971 1923 4050 Paenibacillus elgii B69 3761 16828357010164/ 357010163/ 357010162/ 357010161/ 357010160/ 357010159/ 52265810 949 3089 2038 4155 Pantoea sp. GM01 181180 196279 398800469/398800470/ 398800471/ 398800472/ 398800475/ 398800476/ 5153 5747 8713008 1960 4083 Parabacteroides sp. D25 349829 371928 410103031/410103030/ 410103029/ 410103028/ 410103021/ 410103020/ 5146 5741 8643001 1953 4078 Pectobacterium 12026 27179 421082308/ 421082307/421082306/ 421082305/ 421082302/ 421082301/ wasabiae CFBP 3304 5112 5713829 2965 1918 4045 Pseudanabaena biceps 21843 38743 443475022/443475021/ 443475020/ 443475017/ 443475015/ 443475014/ PCC 7429 51355733 853 2990 1942 4068 Pseudoalteromona 80528 95485 409203052/409203051/ 409203050/ 409203049/ 409203047/ 409203046/ 5238 5821 9613100 2050 4165 Pseudoalteromonas 7985 26765 442610065/ 442610064/442610063/ 442610062/ 442610058/ 442610057/ luteoviolacea B = 5217 5802939 3079 2028 4145 ATCC 29581 Pseudomonas mandelii 1337696 1354409407366557/ 407366556/ 407366555/ missing 407366548/ 407366547/ JR-1 52285813 951 2040 4157 Pseudomonas 141817 157158 359780192/ 359780191/359780190/ missed by 359780188/ 359780187/ psychrotolerans L19 5173 5765892 ORF finder 1981 4104 tool Pseudomonas syringae 83306 107608470894104/ 470894103/ 470894102/ 470894100/ 470894093/ 470894092/ Lz4W5133 5731 851 2988 1940 4066 Roseobacter sp. 2039740 2052609 86139888/86139887/ 86139886/ 86139885/ 86139884/ 86139883/ MED 193 5224 5808 9473087 2036 4153 Ruminococcaceae 39789 54066 332655416/ 332655415/332655414/ 332655413/ 332655411/ 332655410/ bacterium D16 5114 5715 8312968 1920 4047 Salmonella enterica 4758712 4773847 375004441/ 375004440/375004439/ 375004438/ 375004435/ 375004434/ subsp. enterica serovar 51115712 828 2964 1917 4044 Infanti Salmonella enterica 116457 131625422028739/ 422028738/ 422028737/ 422028736/ 422028733/ 422028732/serovar Typhimurium 5158 5751 876 3014 1965 4088 STm1 uid181283Salmonella enterica 348410 363578 458765354/ 458765353/ 458765352/458765351/ 458765348/ 458765347/ serovar Typhimurium 5170 5762 889 30281978 4101 ST1660 06 uid190371 Salmonella enterica 28160 43328 422033790/422033789/ 422033788/ 422033787/ 422033784/ 422033783/ serovarTyphimurium 5177 5769 897 3035 1986 4108 STm2 uid181284 Salmonellaenterica 30253 45421 427597642/ 427597641/ 427597640/ 427597639/427597636/ 427597635/ serovar Typhimurium 5215 5800 937 3077 2026 4144STm3 uid181357 Salmonella enterica 28155 43323 427557943/ 427557942/427557941/ 427557940/ 427557937/ 427557936/ serovar Typhimurium 52345817 957 3096 2046 4162 STm8 uid181355 Salmonella enterica 30250 45412427682142/ 427682141/ 427682140/ 427682139/ 427682136/ 427682135/serovar Typhimurium 5251 5830 975 3115 2064 4179 STm12 uid181362Salmonella enterica 28233 43401 427622026/ 427622025/ 427622024/427622023/ 427622020/ 427622019/ serovar Typhimurium 5256 5835 980 31222069 4182 STm4 uid181358 Salmonella enterica 28172 43334 427576011/427576010/ 427576009/ 427576008/ 427576005/ 427576004/ serovarTyphimurium 5260 5837 984 3126 2073 4186 STm9 uid181356 Salmonellaenterica 28220 43388 427658747/ 427658746/ 427658745/ 427658744/427658741/ 427658740/ serovar Typhimurium 5274 5847 1000 3143 2089 4201STm11 uid181361 Salmonella enterica 28164 42133 missing missing427664029/ 427664028/ 427664025/ 427664024/ serovar Typhimurium 11753149 2094 4206 STm11 uid181361 Salmonella enterica 105150 120318167991286/ 167991285/ 167991284/ 167991283/ 167991280 167991279/ subsp.enterica serovar 5247 5828 971 3110 4175 4,[5],12:i:-str. CVM23701Selenomonas sputigena 547454 560116 260887941/ 260887940/ 260887939/260887938/ 260887934/ truncated ATCC 35185 5120 5721 837 2975 1926Shewanella baltica 211735 229091 418024977/ 418024978/ 418024979/418024983/ 418024984/ 418024985/ OS625 5122 5723 839 2977 1928 4054Sphingobium indicum 80766 95394 390169179/ 390169178/ 390169177/390169174/ 390169173/ 390169172/ B90A 5268 5843 993 3136 2082 4194Sporolactobacillus 5785 18771 404330921/ 404330920/ 404330919/404330918/ 404330917/ 404330916/ vineae DSM 21990 = 5413 5845 997 31402086 4198 SL153 Sporosarcina 827385 843078 340357063/ 340357064/340357065/ 340357066/ 340357070/ 340357071/ newyorkensis 2681 5206 5795927 3067 2016 4135 Stomatobaculum 64338 77737 373106083/ 373106084/373106085/ 373106086/ 373106087/ 373106088/ longum 5410 5946 1173 32791905 4034 (Lachnospiraceae bacterium ACC2) Stomatobaculum 21037 34636373106631/ 373106630/ 373106629/ 373106628/ 373106623/ Missing longum5412 5947 1174 3280 1906 (Lachnospiraceae bacterium ACC2) Streptomycessp. 3417840 3430694 302520235/ 302520234/ 302520233/ 302520232/302520231/ 302520230/ SPB78 5273 5846 999 3142 2088 4200 Thiorhodovibriosp. 970 1742073 1757839 381159244/ Thi970DRAFT_(—) 381159246/Thi970DRAFT_(—) 381159257/ 381159258/ 5286 2972/5701 1018 2976/6182 21034214 Thiothrix nivea DSM 4299231 4317524 386818309/ 386818310/386818311/ 386818316/ 386818318/ 386818319/ 5205 5197 5787 918 3058 20074127 Vibrio cholerae 4260B 149276 164412 440708890/ 440708891/440708892/ 440708895/ 440708896/ 440708897/ 5229 5814 952 3091 2041 4158Vibrio cholerae B33 48656 63703 229509128/ 229509127/ 229509126/229509124/ 229509123/ 229509122/ 5205 5794 926 3066 2015 4134 Vibriocholerae B33 82818 97865 153821444/ 153821461/ 153821425/ 153821428/153821427/ 153821414/ 5219 5805 942 3082 2031 4148 Vibrio cholerae 747220636 421334488/ missing 421334490/ 421334491/ 421334492/ 421334493/CP1048(21) 5245 968 3107 2057 4172 Vibrio cholerae 4634 17819 424605792/missing 424605794/ 424605795/ 424605796/ 424605797/ CP1050(23) 5210 9313071 2020 4139 Vibrio cholerae H1 310730 323915 457927364/ missing457927366/ 457927367/ 457927368/ 457927369/ 5155 873 3010 1962 4085Vibrio cholerae HC- 4700 17885 423730156/ missing 423730158/ 423730159/423730160/ 423730161/ 17A1 5136 854 2991 1943 4069 Vibrio cholerae HC-4629 17814 424001206/ missing 424001208/ 424001209/ 424001210/424001211/ 17A2 5145 863 3000 1952 4077 Vibrio cholerae HC- 4649 17834423144201/ missing 423144203/ 423144204/ 423144205/ 423144206/ 19A1 5185905 3044 1994 4115 Vibrio cholerae HC- 4631 17816 424005362/ missing424005364/ 424005365/ 424005366/ 424005367/ 37A1 5166 884 3023 1973 4096Vibrio cholerae HC- 4635 17820 422924882/ missing 422924884/ 422924885/422924886/ 422924887/ 38A1 5248 972 3111 2061 4176 Vibrio cholerae HC-4623 17808 424609627/ missing 424609629/ 424609630/ 424609631/424609632/ 39A1 5263 988 3131 2077 4189 Vibrio cholerae HC- 4650 17814424612431/ missing 424612433/ 424612434/ 424612435/ 424612436/ 41A1 5208929 3069 2018 4137 Vibrio cholerae HC- 4886 18071 418348066/ missing418348068/ 418348069/ 418348070/ 418348071/ 43A1 5259 983 3125 2072 4185Vibrio cholerae HC- 7242 20406 421345611/ missing 421345612/ 421345553/421345630/ 421345651/ 46A1 5241 964 3103 2053 4168 Vibrio cholerae HC-298 12976 422901578/ missing 422901580/ 422901581/ 422901582/ 422901583/48A1 5222 945 3085 2034 4151 Vibrio cholerae HC- 7286 20471 417812640/missing 417812642/ 417812643/ 417812644/ 417812645/ 49A2 5242 965 31042054 4169 Vibrio cholerae HC- 4633 17818 424644165/ missing 424644167/424644168/ 424644169/ 424644170/ 56A2 5270 995 3138 2084 4196 Vibriocholerae HC- 4631 17816 424651808/ missing 424651810/ 424651811/424651812/ 424651813/ 57A2 5230 953 3092 2042 4159 Vibrio cholerae HC-6230 19394 418354574/ missing 418354555/ 418354534/ 418354592/418354501/ 61A1 5167 885 3024 1974 4097 Vibrio cholerae HC- 4643 17828423891874/ missing 423891876/ 423891877/ 423891878/ 423891879/ 62A1 5249973 3112 2062 4177 Vibrio cholerae HC- 4643 17828 424023371/ missing424023373/ 424023374/ 424023375/ 424023376/ 62B1 5207 928 3068 2017 4136Vibrio cholerae HC- 4641 17826 443502702/ missing 443502704/ 443502705/443502706/ 443502707/ 64A1 5269 994 3137 2083 4195 Vibrio cholerae HC-4643 17828 443506617/ missing 443506619/ 443506620/ 443506621/443506622/ 65A1 5262 987 3130 2076 4188 Vibrio cholerae HC- 4639 17824443510722/ missing 443510724/ 443510725/ 443510726/ 443510727/ 67A1 5127844 2981 1933 4059 Vibrio cholerae HC- 4637 17822 424026175/ missing424026177/ 424026178/ 424026179/ 424026180/ 69A1 5129 846 2983 1935 4061Vibrio cholerae HC- 4639 17824 422905802/ missing 422905804/ 422905805/422905806/ 422905807/ 70A1 5142 860 2997 1949 4074 Vibrio cholerae HC-4639 17824 443522965/ missing 443522967/ 443522968/ 443522969/443522970/ 72A2 5212 933 3073 2022 4140 Vibrio cholerae HC- 4635 17820423926648/ missing 423926650/ 423926651/ 423926652/ 423926653/ 77A1 5202923 3063 2012 4131 Vibrio cholerae HC- 312355 325519 443530594/ missing443530596/ 443530597/ 443530598/ 443530599/ 7A1 5209 930 3070 2019 4138Vibrio cholerae HC- 7298 20483 443537943/ missing 443537945/ 443537946/443537947/ 443537948/ 81A1 5130 847 2984 1936 4062 Vibrio cholerae HFU-4640 17825 422912398/ missing 422912400/ 422912401/ 422932402/422912403/ 02 5178 898 3036 1987 4109 Vibrio cholerae O1 str. 4571 17756458013689/ missing 458013691/ 458013692/ 458013693/ 458013694/2010EL-1792 5176 896 3034 1985 4107 Vibrio cholerae O1 str. 116442118493 458067976/ 458067975/ 458067974/ 458067973/ 458067972/ 458067971/3582-05 5402 6206 1171 3277 2249 4029 Vibrio cholerae O1 str. 4667 17831472149945/ missing 472149947/ 472149948/ 472149949/ 472149950/ EC-00095266 991 3134 2080 4192 Vibrio cholerae O1 str. 4644 17829 472157764/missing 472157766/ 472157767/ 472157768/ 472157769/ EC-0027 5272 9983141 2087 4199 Vibrio cholerae O1 str. 4648 17833 472166840/ missing472166842/ 472166843/ 472166844/ 472166845/ EDC-020 5258 982 3124 20714184 Vibrio cholerae O1 str. 119978 135025 449054089/ 449054088/449054087/ 449054085/ 449054084/ 449054083/ Inaba G4222 5171 5763 8903029 1979 4102 Vibrio cholerae O1 str. 4644 17829 472214605/ missing472214607/ 472214608/ 472214609/ 472214610/ Nep-21106 5221 944 3084 20334150 Vibrio cholerae Ol str. 4644 17829 472217724/ missing 472217726/472217727/ 472217728/ 472217729/ Nep-21113 5149 867 3004 1956 4080Vibrio cholerae O1 str. 4650 17835 472199358/ missing 472199360/472199361/ 472199362/ 472199363/ NHCC-004A 5123 840 2978 1929 4055Vibrio cholerae O1 str. 4648 17833 472210699/ missing 472210701/472210702/ 472210703/ 472210704/ NHCC-010F 5231 954 3093 2043 4160Vibrio cholerae O1 str. 4652 17837 472222753/ missing 472222755/472222756/ 472222757/ 472222758/ PCS-023 5108 825 2961 1913 4042 Vibriocholerae 4335 19471 458152045/ 458152046/ 458152047/ 458152050/458152051/ 458152052/ VC4370 5103 5704 819 2955 1907 4037 Vibrio harveyiCAIM 2186 21054 472443545/ 472443546/ 472443547/ 472443548/ 472443552/472443553/ 1792 5104 5705 820 2956 1908 4038 Vibrio shilonii AK1 4203659655 149189598/ 149189597/ 149189596/ 149189595/ 149189592/ 149189591/5213 5798 935 3075 2024 4142 Vibrio tubiashii ATCC 78266 91498343503397/ 343503398/ 343503399/ 343503400/ 343503401/ 343503402/ 191095257 5836 981 3123 2070 4183 Vibrio cholerae O1 str. 116441 129626458790585/ missing 458790583/ 458790582/ 458790581/ 458790580/2010EL-1798 5137 855 2992 1944 4070 Yersinia ruckeri ATCC 13923 29006238756189/ 238756190/ 238756191/ 238756192/ 238756193/ 238756194/ 294735152 5746 870 3007 1959 4082 Thioalkalivibrio sp. 1195163 1212198289208308/ 289208309/ TK90_1129/ 289208312/ 289208315/ 289208316/ K90mix6225 6227 6229 6231 6233 6235 *Numbers are presented by AccessionNO./SEQ ID NO.

TABLE 11 BREX type 5 systems Genomic Genomic Start End Organism PointPoint brxA* brxB* BrxC/pglY* pglX* pglZ* brxHII* Halopiger xanaduensisSH6 275443 305942 336252375/ 238756192/ 336252376/ 336252387/ 336252390/336252392/ uid68105 5065 3007 736 2917 1835 3498 336252378/ 737Halorhabdus utahensis DSM 1919731 1943689 missing 238756192/ 257052978/257052985/ 257052987/ 257052989/ 12940 uid59189 3008 738 2918 1836 3499257052980/ 739 Halobacterium salinarum R1 217533 239329 169237558/238756192/ 169237555/ 169237551/ 169237549/ missing uid61571 5066 3009740 2919 1837 169237557/ 741 Halorubrum lacusprofundi 421881 464896222476096/ 238756192/ 222476093/ 222476090/ 222476089/ 222476088/ ATCC49239 uid58807 5067 3010 742 2920 1838 3500 222476095/ 222476117/ 7432921 Haloarcula hispanica ATCC 401471 426563 344209887/ 238756192/344209884/ 344209878/ 344209879/ 344209877/ 33960 uid72475 5068 3011 7442922 1839 3501 344209886/ 344209881/ 745 2923 halophilic archaeon DL31245839 265734 345007035/ 238756192/ 345007036/ 345007041/ 345007043/345007044/ uid72619 5069 3012 746 2924 1840 3502 345007038/ 747Natrinema pellirubrum DSM 247457 267369 433593286/ 238756192/ 433593287/433593291/ 433593294/ 433593295/ 15624 uid74437 5070 3013 748 2925 18413503 433593289/ 433593292/ 749 2926 Natronorubrum tibetense 16416 35521448302571/ 238756192/ 448302568/ missing 448302567/ 448302575/ GA33 53683014 1111 2193 3527 448302570/ 1112 Haloarcula argentinensis 133870160866 448682081/ 238756192/ 448682078/ 448682072/ 448682071/ 448682070/DSM 12282 5369 3015 1113 3252 2194 3528 448682080/ 1114 Halosimplexcarlsbadense 2- 45427 67411 445667054/ 238756192/ 448413236/ missing448413226/ 448413225/ 9-1 6167 3016 1115 2195 3529 448413238/ 1116*Numbers are presented by Accession NO./SEQ ID NO.

TABLE 12 BREX type 6 systems Genomic Genomic Start End Organism PointPoint brxE* brxA* brxB* Planctomyces limnophilus 3979304 3995634296123323/ 296123322/ 296123321/ DSM 3776 uid48643 6037 5071 5694Anaeromyxobacter 1284321 1301040 220916259/ 220916260/ 220916261/dehalogenans 2CP 1 6038 5072 5695 uid58989 Haliangium ochraceum 798493815906 262193918/ 262193919/ 262193920/ DSM 14365 uid41425 6039 50735696 Haliangium ochraceum 1611313 1628687 262194477/ 262194478/262194479/ DSM 14365 uid41425 6040 5074 5697 Rhodopirellula 94858 111243470888969/ 470888968/ 470888967/ sp. SWK7 6035 5370 5702 Pseudanabaenabiceps 10824 29281 443475075/ 443475074/ 443475073/ PCC 7429 6036 53715943 Organism BrxC/pglY* pglX* pglZ* brxD* brxHI* Planctomyceslimnophilus 296123320/ 296123319/ 296123318/ 296123317/ 296123316/ DSM3776 uid48643 750 2927 1842 4441 3622 Anaeromyxobacter 220916262/220916263/ 220916264/ 220916266/ 220916267/ dehalogenans 2CP 1 751 29281843 4442 3623 uid58989 Haliangium ochraceum 262193921/ 262193922/262193923/ 262193924/ 262193925/ DSM 14365 uid41425 752 2929 1844 44433624 Haliangium ochraceum 262194480/ 262194481/ 262194482/ 262194483/262194484/ DSM 14365 uid41425 753 2930 1845 4444 3625 Rhodopirellula470888966/ 470888965/ 470888964/ 470888963/ 470888962/ sp. SWK7 11176186 2196 4464 3626 Pseudanabaena biceps 443475072/ 443475069/443475065/ 443475063/ 443475062/ PCC 7429 1118 6188 2197 4465 3627*Numbers are presented by Accession NO./SEQ ID NO.

TABLE 13 BREX type 3 systems Genomic Genomic Start End Organism PointPoint brxF* brxC/pglY* pglXI* brxHII* pglZ* brxA* Acetohalobiumarabaticum 1471809 1481230 302392145/ 302392144/ 302392143/ missing302392142/ 302392141/ DSM 5501 uid51423 5919 754 3343 1846 5075Acidothermus 895934 911157 117928019/ 117928020/ 117928022/ 117928024/117928025/ 117928026/ cellulolyticus 11B 5980 755 3344 3504 1847 5076uid58501 Anaerobaculum mobile 1894751 1909131 392408180/ 392408179/392408178/ 392408176/ 392408175/ 392408174/ DSM 13181 uid168323 5981 7563345 3505 1848 5077 Bacteroides vulgatus 4615002 4627784 missing150006230/ 150006231/ 150006233/ 150006234/ 150006235/ ATCC 8482uid58253 757 3346 3506 1849 5078 Caldicellulosiruptor 671655 683698312792856/ 312792857/ 312792858/ Calkr_0625/ 312792859/ 312792860/kristjanssonii 177R1B 5982 758 3347 6173 1850 5079 uid60393 Chloroflexusaggregans 1376256 1391910 219848031/ 219848033/ 219848032/ 219848036/219848037/ 219848038/ DSM 9485 uid58621 5983 759 3348 3507 1853 5080219848035/ 3349 Desulfovibrio aespoeensis 1822100 1838149 317153363/317153364/ 317353366/ 317153368/ 317153369/ 317153370/ Aspo 2 uid426135984 760 3350 3508 1852 5081 Dichelobacter nodosus 185801 200163146328698/ 146329396/ 146329329/ 146329165/ 146329856/ 146328877/VCS1703A uid57643 5985 761 3351 3509 1853 5082 Methanocaldococcus 892177917259 289192374/ 289192375/ 289192376/ missing 289192377/ 289192378/FS406 22 uid42499 5986 762 3352 1854 5083 Methanosalsum zhilinae 13988671423843 336477236/ 336477235/ 336477234/ 336477230/ 336477229/336477228/ DSM 4017 uid68249 5987 764 3353 3510 1855 5084 336477237/3354 Methylacidiphilum 315550 333912 189218342/ 189218341/ 189218335/189218338/ 189218337/ 189218336/ infernorum V4 uid59161 5988 765 33553511 1856 5085 189218340/ 3356 Nitrosococcus oceani 53559 7030777163602/ 77163603/ 77163606/ 77163610/ 77163611/ 77163612/ ATCC 19707uid58403 5990 767 3358 3513 1858 5087 Nitrosococcus watsonii C 4370358760 300112781/ 300112782/ 300112784/ 300112788/ 300112789/ 300112790/113 uid50331 5991 768 3359 3514 1859 5088 Parvibaculum 1304571 1320948154251635/ 154251634/ 154251631/ 154251630/ 154251629/ 154251628/lavamentivorans DS 1 5992 769 3360 3515 1860 5089 uid58739 Parvibaculum3796620 3812997 154253985/ 154253986/ 154253989/ 154253990/ 154253991/154253992/ lavamentivorans DS 1 5993 770 3361 3516 1861 5090 uid58739Planctomyces brasiliensis 1319340 1355964 325107713/ 325107712/325107710/ 325107709/ 325107708/ 325107707/ DSM 5305 uid60583 5994 7713362 3517 1862 5091 Syntrophothermus 1307414 1328282 297617453/297617452/ 297617451/ missing 297617450/ 297617449/ lipocalidus DSM12680 5995 772 3363 1863 5092 uid49527 Tepidanaerobacter 1934820 1944222438003074/ 438003073/ 438003072/ missing 438003071/ 438003070/acetatoxydans Re1 5996 774 3364 1864 5093 uid184827 TepidanaerobacterRe1 1933717 1943118 332799810/ 332799809/ 332799808/ missing 332799807/332799806/ uid66873 5997 775 3365 1865 5094 Thermanaerovibrio 15518231568220 269793100/ 269793099/ 269793097/ 269793095/ 269793094/269793093/ acidaminovorans DSM 5998 776 3366 3518 1866 5095 6589uid41925 Thermoanaerobacter 974096 986151 320115754/ 320115755/320115756/ 320115757/ 320115758/ 320115759/ brockii finnii Ako 1 5999777 3367 3519 1867 5096 uid55639 Thermoanaerobacter 1369765 1379419289578546/ 289578545/ 289578544/ missing 289578542/ 289578541/ italicusAb9 uid46241 6000 778 3368 1868 5097 Thermoanaerobacter 981903 993958167037338/ 167037339/ 167037340/ 167037341/ 167037342/ 167037343/pseudethanolicus ATCC 6001 779 3369 3520 1869 5098 33223 uid58339Thermoanaerobacterium 2325486 2337569 390935378/ 390935377/ 390935376/390935375/ 390935374/ 390935373/ saccharolyticum JW SL 6002 780 33703521 1870 5099 YS485 uid167781 Thermoanaerobacterium 1846040 1858147433655487/ 433655486/ 433655485/ 433655484/ 433655483/ 433655482/thermosaccharolyticum 6003 781 3371 3522 1871 5100 M0795 uid184821Thermoanaerobacterium 1036830 1048931 333896805/ 333896806/ 333896807/333896808/ 333896809/ 333896810/ xylanolyticum LX 11 6004 782 3372 35231872 5101 uid63163 Pelotomaculum 698030 719373 147677041/ 147677050/#N/A 147677052/ thermopropionicum SI 616 3403 1177 uid58877planctomycete KSU-1 302255 318802 386811047/ 386811046/ 386811042/386811038/ 386811037/ 386811036/ 6005 1119 3373 3530 2198 5372 Bacilluscereus W 64027 76920 196035241/ 196035162/ 196035235/ 196035256/196035132/ 196035248/ 6006 1120 3374 3531 2199 5373 Clostridiumthermocellum 67367 77609 missing 419725844/ 419725843/ 419725842/419725841/ 419725839/ YS 1121 3375 3532 2200 5374 419725840/ 5375Lachnospiraceae bacteriu 835791 847349 336426893/ 336426892/ 336426890/336426889/ 336426888/ 336426887/ 6007 1122 3376 3533 2201 5376336426891/ 3377 Enterococcus faecium 115722 257885933/ 257885934/257885935/ missing 257885936/ 257885937/ 1,231,501 6008 1123 3378 22026170 Caloramator australicus 17570 34190 397905665/ 397905666/397905667/ 397905670/ 397905671/ 397905672/ RC3]Length = 15 6009 11243379 3534 2203 5377 397905668/ 3380 397905669/ 3381 397905678/ 3382Pseudoalteromonas marina 51753 65055 392538886/ 392538885/ 392538884/392538883/ 392538882/ 392538880/ mano4 6010 1125 3383 3535 2204 5378392538881/ 5379 Desulfotomaculum 104421 114874 323701233/ 323701234/323701235/ missing 323701238/ 323701239/ nigrificans DSM 574 6011 11263384 2205 5380 Dethiosulfovibrio 2471763 2489488 288575103/ 288575102/288575100/ DpepDRAFT_(—) 288575095/ 288575094/ peptidovorans DSM 110026012 1127 3385 2372/6176 2206 5381 Kingella denitrificans 998416 1009424325266722/ 325266723/ 325266724/ 325266725/ 325266726/ 325266727/ ATCC33394 6013 1128 3386 6177 2207 5382 Alcanivorax 66342 85539 408373946/408373945/ 408373941/ 408373939/ 408373938/ 408373937/ hongdengensisA-11-3 6014 1129 3387 3536 2208 5383 Bacillus cereus BAG2X1- 41344464144054 423398560/ 423398559/ 423398558/ 401647033/ 423398557/423398556/ 1 6015 1130 3388 6178 2209 5384 Nitrosococcus oceani 570876587579 254435536/ 254435628/ 254435024/ missing 254436183/ 254435793/AFC27 6016 1131 3389 2210 5385 Methyloversatilis 13404 25532 missing334130736/ 334130735/ 334130734/ 334130733/ 334130732/ universalis FAM51132 3390 3537 2211 5386 Thermoplasmatales 1730 14243 472439489/472439490/ 472439492/ missing 472439494/ 472439495/ archaeon SCGCAB-539- 6017 1133 3391 2212 5387 N05 Pseudomonas sp. GM55 71736 85194398889060/ 398889059/ 398889058/ PMI31_00817 398889056/ 398889055/ 60181134 3392 2213 5388 Bacillus cereus 03BB108 37106 46755 196044400/196044278/ 196044173/ 168166142 196044220/ 196044133/ 6019 1135 33932214 5389 Treponema primitia ZAS- 9000 34287 374812240/ 374812241/374812242/ 374812236/ 374812248/ 374812249/ 1 6020 1136 3394 3538 22155415 Bacillus methanolicus 39939 49733 415887046/ 415887047/ 415887048/MGA3_16723/ 415887049/ 415887050/ MGA3 6021 1137 3395 6180 2216 5390Vibrio scophthalmi LMG 609 13898 343509541/ 343509540/ 343509539/343509538/ 343509537/ 343509534/ 19158 6022 1138 3396 3539 2217 5391343509535/ 5392 343509536/ 5393 Thiorhodovibrio sp. 970 32332 48989381156867/ 381156866/ 381156864/ Thi970DRAFT_(—) 381156859/ 381156858/6023 1139 3397 0448/6183 2218 5394 Ectothiorhodospir 1 13805 374623711/374623710/ 374623709/ 374623707/ 374623706/ missing 6024 1140 3398 35402219 Clostridium papyrosolvens 27432 39463 326204497/ 326204498/326204499/ 326204500/ 326204501/ 326204502/ DSM 2782 6025 1141 3399 35412220 5395 Thermoanaerobacter 35382 49116 326389944/ 326389943/326389941/ 326389940/ 326389939/ 326389938/ ethanolicus JW 200 6026 11423400 3542 2221 5396 Thermoanaerobacter 8992 18633 256752453/ 256752454/256752456/ missing 256752458/ 256752459/ ethanolicus CCSD1 6027 11433401 2222 5397 256752455/ 1144 Nitrococcus mobilis Nb- 2181486 219978288811251/ 88811250/ 88811247/ NB231_10583/ 88811242/ 88811241/ 231 60281145 3402 6184 2223 5398 *Numbers are presented by Accession NO./SEQ IDNO.

TABLE 14 BREX type 2 systems Genomic Genomic Start End Organism PointPoint pglW* pglX* pglY* pglZ* brxD* brxHI* Burkholderia thailandensis131918 145741 83720701/ 83719870/ 83719623/ 83719262/ missing missingE264 uid58081 6091 2931 783 1873 Candidatus Accumulibacter 38695503890263 257094932/ 257094931/ 257094926/ 257094925/ 257094924/257094923/ phosphalis clade IIA UW 1 6092 2932 784 1874 4445 3596uid59207 Corallococcus coralloides 6591319 6611764 383457207/ 383457206/383457202/ 383457203/ missing 383457201/ DSM 2259 uid157997 6093 2933785 1875 3597 383457204/ 786 Corynebacterium variabile 1913373 1933608340794635/ 340794636/ 340794637/ 340794638/ 340794639/ 340794640/ DSM44702 uid62003 6094 2934 787 1876 4446 3598 Frankia CcI3 uid583973489708 3507585 86741639/ 86741640/ 86741641/ 86741642/ 86741643/86741644/ 6095 2935 788 1877 4447 3599 Frankia EuI1c uid42615 69519046971263 312199458/ 312199459/ 312199460/ 312199461/ 312199462/312199463/ 6096 2936 789 1878 4448 3600 Haliangium ochraceum DSM 15651701582937 262194452/ 262194453/ 262194454/ 262194455/ 262194456/262194457/ 14365 uid41425 6097 2937 790 1879 791 3601 Microlunatusphosphovorus 3075705 3093432 336118511/ 336118510/ 336118509/ 336118508/336118507/ 336118506/ NM 1 uid68055 6098 2938 792 1880 4449 3602Micromonospora aurantiaca 1329830 1350410 302865792/ 302865793/302865796/ 302865797/ 302865798/ 302865799/ ATCC 27029 uid42501 60992939 793 1881 4450 3603 Mycobacterium gilvum PYR 3386977 3404461145223791/ 145223790/ 145223789/ 145223788/ 145223787/ 145223786/ GCKuid59421 6100 2940 794 1882 4451 3604 Nocardia cyriacigeorgica 19749631993641 379708002/ 379708001/ 379708000/ 379707999/ 379707998/379707997/ GUH 2 uid89395 6101 2941 795 1883 4452 3605 Polaromonas170793 210985 121582867/ 121582865/ 121582862/ 121582861/ 121582860/121582859/ naphthalenivorans CJ2 6102 2942 796 1884 4453 3606 uid58273121582883/ 2943 Saccharomonospora viridis 508003 530821 257054581/257054590/ 257054591/ 257054592/ 257054593/ 257054594/ DSM 43017uid59055 6103 2944 797 1885 4454 3607 Saccharopolyspora erythraea5714083 5731753 134101641/ 134101640/ 134101639/ 134101638/ 134101637/134101636/ NRRL 2338 uid62947 6104 2945 798 1886 4455 3608 Saccharothrixespanaensis 1882970 1907934 433603667/ 433603658/ 433603653/ 433603652/433603651/ 433603650/ DSM 44229 uid184826 6105 2946 799 1887 4456 3609Singulisphaera acidiphila 2421532 2440518 430742885/ 430742884/430742881/ 430742880/ 430742879/ 430742878/ DSM 18658 uid81777 6106 2947800 1888 4457 3610 Sorangium cellulosum So ce 10676864 10731426162455957/ 162455958/ 162455964/ 162455967/ 162455968/ 162455969/ 56uid61629 6107 2948 801 1889 4458 3611 162455970/ 6108 162455983/ 6109Streptomyces coelicolor A3 2 7348537 7376403 21224924/ 32141309/21224932/ 21224933/ 21224936/ 21224937/ uid57801 6110 2949 802 1890 44593612 Streptomyces griseus NBRC 1877109 1900853 182435393/ 182435394/182435399/ 182435400/ 182435403/ 182435404/ 13350 uid58983 6111 2950 8031891 4460 3613 Thermobifida fusca YX 810381 853382 72161105/ 72161128/72161113/ 72161114/ 72161115/ 72161116/ uid57703 6112 2951 804 1892 44613614 Thermobispora bispora DSM 1764882 1779583 296269520/ 296269521/296269522/ 296269523/ missing missing 43833 uid48999 6113 2952 805 1893Hahella chejuensis KCTC 3587257 3606877 83646207/ 83646204/ 83646203/83646202/ 83646201/ 83646200/ 2396 uid58483 6114 2953 806 1894 4462 3615Saccharomonospora glauca 539845 562776 384564483/ 384564490/ 384564491/384564492/ 384564493/ 384564494/ K62 6115 3253 1146 2224 4466 3616Rhodococcus triatomae BKS 58688 79518 453074717/ 453074718/ 453074722/453074723/ 453074724/ 453074725/ 15-14 6116 3254 1147 2225 4467 3617Saccharomonospora cyanea 604367 625835 375098936/ 375098940/ 375098941/375098942/ 375098943/ 375098944/ NA-134 6117 3255 1148 2226 4468 3618Gordonia polyisoprenivorans 51878 69760 359765504/ 359765505/ 359765506/359765507/ 359765508/ 359765509/ NBRC 16320 6118 3256 1149 2227 44693619 Amycolatopsis azurea DSM 30190 53418 451335443/ 451335436/451335435/ 451335434/ 451335432/ 451335431/ 43854 6119 3257 1150 22284470 3620 Marinobacter sp. ELB17 49994 72968 126666550/ 126666547/126666537/ 126666536/ 126666535/ 126666534/ 6120 3258 1151 2229 44713621 Saccharopolyspora erythraea 144893 160200 291003191/ 291003192/291003193/ 291003194/ 291003195/ missing NRRL 2338 6121 3259 1152 22304472 Streptomyces turgidiscabies 9173 41654 440700787/ 440700766/440700790/ 440700796/ 440700764/ 440700770/ Car8 6122 3260 1153 22314473 3628 Gemma taobscuriglobus 27609 48046 168697904/ 168697902/168697899/ 168697898/ 168697897/ 168697896/ UQM 2246 6123 3261 1154 22324474 3629 Rhodococcus ruber BKS 20- 6599 29593 458780602/ 458780601/458780598/ 458780597/ 458780596/ 458780593/ 38 6124 3262 1155 2233 44753630 Mycobacterium 216677 234966 383824736/ 383824739/ 383824740/383824741/ 383824742/ 383824743/ xenopi RIVM700367 6125 3263 1156 22344476 3631 Micromonospora sp. ATCC 4955630 4982819 238063210/ 238063211/238063221/ 238063222/ 238063223/ 238063224/ 39149 6126 3264 1157 22354477 3632 Saccharomonospora 1696320 1718030 383828993/ 383828987/383828986/ 383828985/ 383828984/ 383828983/ xinjiangensis XJ-54 61273265 1158 2236 4478 3633 Bradyrhizobium sp. ORS 375 19316 37325365878962/ 365878961/ 365878960/ 365878959/ 365878958/ 365878957/ 61283266 1159 2237 4479 3634 Burkholderia thailandensis 3406043 3419866257140682/ 257140680/ 257140679/ 257140678/ missing missing E264 61293267 1160 2238 Planctomyces maris DSM 100676 119360 149176313/149176311/ 149176310/ 149176309/ 149176308/ 149176307/ 8797 6130 32681161 2239 4480 3635 Streptomyces gancidicus BKS 67474 97086 458859650/458859651/ 458859660/ 458859661/ 458859664/ 458859665/ 13-15 6131 32691162 2240 4481 3636 Gordonia amicalis NBRC 3958 21621 441515888/441515889/ 441515890/ 441515891/ 441515892/ 441515893/ 100051 = JCM11271 6132 3270 1163 2241 4482 3637 Mycobacterium intracellulare 1496330468 254821509/ 254821510/ 254821511/ 254821513/ 254821514/ missingATCC 13950 6133 3271 1164 2242 4483 Phaeospirillum molischianum 972729750 381168760/ 381168763/ 381168766/ 381168767/ 381168768/ 381168769/DSM 120 6134 3272 1165 2243 4484 3638 Nitrococcus mobilis Nb-231 32846583304962 88810583/ 88810586/ 88810589/ 88810590/ 88810591/ 88810592/ 61353273 1166 2244 4485 3639 88810585/ 6136 Frankia sp. EUN1f 4751 24481288920048/ 288920049/ 288920050/ 288920051/ 288920052/ 288920053/ 61373274 1167 2245 4486 3640 Pseudomonas stutzeri NF13 77221 95110452746641/ missing 452746643/ 452746644/ 452746645/ 452746646/ 6138 11682246 4487 3641 Dietzia cinnamea P4 1 10875 missing 319947886/ 319947887/319947888/ 319947889/ 319947890/ 3275 1169 2247 4488 3642 *Numbers arepresented by Accession NO./SEQ ID NO.

TABLE 15 BREX type 4 systems Genomic Genomic Start End Organism PointPoint brxP* brxC/pglY* pglZ* brxL* Aciduliprofundum MAR08 12115351229434 432329235/ 432329234/ 432329233/ 432329232/ 339 uid184407 3428807 1895 4403 Anaerobaculum mobile DSM 1574689 1585597 392407896/392407895/ 392407894/ 392407893/ 13181 uid168323 3429 808 1896 4404392407897/ 3430 Candidatus Desulforudis 837185 861804 169830967/169830968/ 169830970/ 169830971/ audaxviator MP104C 3431 809 1897 4405uid59067 Coprothermobacter 1373786 1384972 206895424/ 206896068/206895399/ 206895834/ proteolyticus DSM 5265 3432 810 1898 4406 uid59253206895920/ 3433 Cyanobacterium stanieri PCC 2847921 2863377 428774403/428774405/ 428774399/ 428774398/ 7202 uid183337 3434 812 1899 4407Denitrovibrio acetiphilus 583467 593592 291286509/ 291286510/ 291286511/291286512/ DSM 12809 uid46657 3435 813 1900 4408 Desulfitobacterium793871 804051 431792781/ 431792782/ 431792783/ 431792784/dichloroeliminans LMG P 3436 814 1901 4409 21439 uid82555 Geobacter M21uid59037 937933 948073 253699433/ 253699434/ 253699435/ 253699436/ 3437815 1902 4410 Prevotella denticola F0289 414128 424374 327312660/327312662/ 327312663/ 327312664/ uid65091 3438 816 1903 4411 Thermotogapetrophila RKU 1747153 1782820 148270868/ 148270869/ 148270870/148270871/ 1 uid58655 3439 817 1904 4412 Thermomicrobium roseum 91162104624 A truncated 221635509/ 221635513/ 221635514/ DSM 5159 uid59341brxP was 1714 1714 4299 missed by ORF finder tool. *Numbers arepresented by Accession NO./SEQ ID NO.

TABLE 16 Summary of distribution of BREX types across genomes BREX BREXBREX BREX BREX BREX Organism #1 #5 #6 #3 #2 #4 Comments 1 Pseudanabaenabiceps PCC 7429 x x Genome contains two BREX systems: #1 and #6 2Moorella thermoacetica ATCC 39073 x uid58051 3 Tepidanaerobacteracetatoxydans Re1 x x Genome contains two BREX uid184827 systems: #1 and#3 4 Tepidanaerobacter Re1 uid66873 x x Genome contains two BREXsystems: #1 and #3 5 Thiorhodovibrio sp. 970 x x Genome contains twoBREX systems: #1 and #3 6 Marinobacter sp. ELB17 x x Genome contains twoBREX systems: #1 and #2 7 Microlunatus phosphovorus NM 1 uid68055 x xGenome contains two BREX systems: #1 and #2 8 Acetivibrio cellulolyticusCD2 x 9 Acidiphilium multivorum AIU301 uid63345 x 10 Acidithiobacillusferrivorans SS3 uid67387 x 11 Acidovorax sp. NO-1 x 12 Acinetobacterbaumannii AB5256 x 13 Acinetobacter baumannii AYE uid61637 x 14Acinetobacter baumannii Naval-18 x 15 Acinetobacter baumannii OIFC098 x16 Acinetobacter baumannii WC-136 x 17 Acinetobacter johnsonii SH046 x18 Acinetobacter junii SH205 x 19 Acinetobacter lwoffii SH145 x 20Acinetobacter radioresistens DSM 6976 = x NBRC 102413 21 Acinetobactersp. P8-3-8 x 22 Acinetobacter sp. SH024 x 23 Actinomyces neuii BVS029A5x 24 Actinomyces sp. ICM47 x 25 Actinomyces sp. oral taxon 178 str x 26Alcanivorax pacificus W11-5 x 27 Alcanivorax sp. DG881 x 28 Alteromonasmacleodii Black Sea 11 x uid176365 29 Anaeromyxobacter dehalogenans 2CPC x uid58135 30 Aromatoleum aromaticum EbN1 uid58231 x 31 Arthrobacternitroguajacolicus Rue61a x uid174511 32 Aurantimonas manganoxydansSI85-9A1 x 33 Azospirillum lipoferum 4B uid82343 x 34 Bacillus cereusBAG6X1-2 x 35 Bacillus cereus H3081.97 x 36 Bacillus cereus HuB2-9 x 37Bacteroides coprophilus DSM 18228 x 38 Bacteroides ovatus CL02T12C04 x39 Bacteroides ovatus SD CC 2a x 40 Bacteroides ovatus SD CMC 3f x 41Bacteroides sp. 1_1_14 x 42 Bacteroides sp. 2_1_7 x 43 Bacteroides sp.3_1_33FAA x 44 Bacteroides sp. D1 x 45 Bacteroides sp. D2 x 46Bacteroides sp. 2_1_22 x 47 Bacteroides xylanisolvens SD CC 1b x 48Bifidobacterium angulatum DSM 20098 x 49 Bifidobacterium animalis ATCC25527 x uid162513 50 Bifidobacterium bifidum IPL x 51 Bifidobacteriumbifidum NCIMB 41171 x 52 Bordetella parapertussis Bpp5 uid177516 x 53Brachybacterium paraconglomeratum LC44 x 54 Brevibacterium mcbrellneriATCC 49030 x 55 Burkholderia CCGE1001 uid42975 x 56 Burkholderiagladioli BSR3 uid66301 x 57 Burkholderia vietnamiensis G4 uid58075 x 58Calditerrivibrio nitroreducens DSM 19672 x uid60821 59 Carboxydothermushydrogenoformans Z x 2901 uid57821 60 Cellvibrio sp. BR x 61 Chlorobiumphaeobacteroides BS1 x uid58131 62 Clostridium butyricum E4 str. BoNT Ex BL5262 63 Clostridium ljungdahlii DSM 13528 x uid50583 64 Clostridiumperfringens C str. JGS1495 x 65 Clostridium perfringens D str. JGS1721 x66 Clostridium sticklandii DSM 519 uid59585 x 67 Clostridium SY8519uid68705 x 68 Collinsella aerofaciens ATCC 25986 x 69 Collinsellaintestinalis DSM 13280 x 70 Collinsella stercoris DSM 13279 x 71Coprobacillus sp. 3_3_56FAA x 72 Coprobacillus sp. 8_2_54BFAA x 73Cupriavidus necator N 1 uid68689 x 74 Cyanothece PCC 8802 uid59143 x 75Cylindrospermopsis raciborskii CS-505 x 76 Dehalococcoides VS uid42393 x77 Dehalogenimonas lykanthroporepellens BL x DC 9 uid48131 78 deltaproteobacterium NaphS2 x 79 Desulfitobacterium hafniense Y51 uid58605 x80 Desulfobacula toluolica Tol2 uid175777 x 81 Desulfomicrobiumbaculatum DSM 4028 x uid59217 82 Desulfonatronospira thiodismutansASO3-1 x 83 Desulfosporosinus meridiei DSM 13257 x uid75097 84Desulfotomaculum gibsoniae DS x 85 Desulfovibrio magneticus RS 1uid59309 x 86 Desulfovibrio vulgaris Hildenborough x uid57645 87Desulfovibrio vulgaris RCH1 uid161961 x 88 Desulfurivibrio alkaliphilusAHT2 uid49487 x 89 Enterobacter cloacae ENHKU01 uid172463 x 90Enterobacter hormaechei ATCC 49162 x 91 Enterobacter sp. SST3 x 92Enterococcus faecalis TX0109 x 93 Enterococcus faecalis TX1302 x 94Enterococcus faecium 509 x 95 Enterococcus faecium 511 x 96 Enterococcusfaecium 514 x 97 Enterococcus faecium C1904 x 98 Enterococcus faeciumC497 x 99 Enterococcus faecium E0679 x 100 Enterococcus faecium E1731 x101 Enterococcus faecium E1904 x 102 Enterococcus faecium E2883 x 103Enterococcus faecium ERV99 x 104 Enterococcus faecium P1123 x 105Enterococcus faecium P1137 x 106 Enterococcus faecium P1139 x 107Enterococcus faecium TX0133A x 108 Enterococcus faecium TX0133a01 x 109Enterococcus faecium TX0133a04 x 110 Enterococcus faecium TX0133B x 111Enterococcus faecium TX0133C x 112 Erwinia Ejp617 uid159955 x 113Erwinia pyrifoliae DSM 12163 uid159693 x 114 Erwinia pyrifoliae Ep1 96uid40659 x 115 Erythrobacter litoralis HTCC2594 uid58299 x 116Escherichia coli clone D i14 uid162049 x 117 Escherichia coli clone D i2uid162047 x 118 Escherichia coli 2534-86 x 119 Escherichia coli 3.3884 x120 Escherichia coli 4.0522 x 121 Escherichia coli 96.154 x 122Escherichia coli B41 x 123 Escherichia coli B799 x 124 Escherichia coliDEC10E x 125 Escherichia coli DEC10F x 126 Escherichia coli DEC13A x 127Escherichia coli DEC13B x 128 Escherichia coli DEC13C x 129 Escherichiacoli DEC13D x 130 Escherichia coli DEC13E x 131 Escherichia coli DEC14Bx 132 Escherichia coli DEC14C x 133 Escherichia coli DEC14D x 134Escherichia coli DEC7B x 135 Escherichia coli DEC8A x 136 Escherichiacoli DEC8B x 137 Escherichia coli DEC9A x 138 Escherichia coli DEC9B x139 Escherichia coli DEC9C x 140 Escherichia coli DEC9D x 141Escherichia coli DEC9E x 142 Escherichia coli HS uid58393 x 143Escherichia coli JB1-95 x 144 Escherichia coli KD2 x 145 Escherichiacoli KTE12 x 146 Escherichia coli KTE139 x 147 Escherichia coli KTE153 x148 Escherichia coli KTE211 x 149 Escherichia coli KTE218 x 150Escherichia coli KTE234 x 151 Escherichia coli KTE47 x 152 Escherichiacoli KTE53 x 153 Escherichia coli KTE6 x 154 Escherichia coli MS 69-1 x155 Escherichia coli O10:K5(L):H4 str. ATCC x 23506 156 Escherichia coliO111 H 11128 uid41023 x 157 Escherichia coli O111:H8 str. CVM9570 x 158Escherichia coli O111:H8 str. CVM9574 x 159 Escherichia coli O111:H8str. CVM9602 x 160 Escherichia coli O111:H8 str. CVM9634 x 161Escherichia coli O113:H21 str. CL-3 x 162 Escherichia coli O25b:ST131str. JIE186 x 163 Escherichia coli OK1180 x 164 Escherichia coli S17 x165 Escherichia coli STEC_94C x 166 Escherichia coli TW10828 x 167Escherichia fergusonii ATCC 35469 x uid59375 168 Eubacteriumcellulosolvens 6 x 169 Exiguobacterium sibiricum 255 15 uid58053 x 170Faecalibacterium prausnitzii A2-165 x 171 Faecalibacterium prausnitziiM21/2 x 172 Flavobacterium branchiophilum FL 15 x uid73421 173Fulvimarina pelagi HTCC2506 x 174 Fusobacterium nucleatum subsp.nucleatum x ATCC 23726 175 Fusobacterium sp. 11_3_2 x 176 Fusobacteriumsp. 2_1_31 x 177 Fusobacterium sp. 3_1_27 x 178 Fusobacterium sp. 3_1_5Rx 179 Fusobacterium sp. 7_1 x 180 Fusobacterium ulcerans ATCC 49185 x181 gamma proteobacterium BDW918 x 182 gamma proteobacterium IMCC3088 x183 Geobacillus sp. G11MC16 x 184 Geobacillus thermoglucosidan x 185Geobacillus WCH70 uid59045 x 186 Geobacter sulfurreducens PCA uid57743 x187 Glaciecola lipolytica E3 x 188 Glaciecola polaris LMG 21857 x 189Glaciecola punicea DSM 14233 = ACAM x 611 190 Haliscomenobacterhydrossis DSM 1100 x uid66777 191 Haloarcula japonica DSM 6131 x 192Halobacillus halophilus DSM 2266 x uid162033 193 Halobacteroideshalobius DSM 5150 x uid184862 194 Halomonas sp. HAL1 x 195 Holdemaniafiliformis DSM 12042 x 196 Holophaga foetida DSM 6591 x 197 Johnsonellaignava ATCC 51276 x 198 Klebsiella oxytoca E718 uid170256 x 199Lachnoanaerobaculum (Eubacterium) x saburreum DSM 3986 200Lachnoanaerobaculum (Eubacterium) x saburreum F0468 201 Lachnospiraceaebacterium 1_1_57FAA x 202 Lachnospiraceae bacterium 8_1_57FAA x 203Lachnospiraceae bacterium oral taxon 082 x str 204 Lachnospiraceae oraltaxon 107 str x 205 Lactobacillus amylovorus GRL1118 x uid160233 206Lactobacillus casei Lpc-37 x 207 Lactobacillus casei UW4 x 208Lactobacillus casei Zhang uid50673 x 209 Lactobacillus helveticus DSM20075 x 210 Lactobacillus helveticus H10 uid162017 x 211 Lactobacillushelveticus R0052 uid174439 x 212 Lactobacillus johnsonii ATCC 33200 x213 Lactobacillus johnsonii FI9785 uid41735 x 214 Lactobacillus reuteriCF48-3A x 215 Lactobacillus reuteri SD2112 uid55357 x 216 Lactobacillusrhamnosus GG uid161983 x 217 Lactobacillus rhamnosus GG uid59313 x 218Lactobacillus rhamnosus LRHMDP2 x 219 Lactobacillus rhamnosus LRHMDP3 x220 Lactobacillus zeae KCTC 3804 x 221 Leuconostoc kimchii IMSNU 11154 xuid48589 222 Magnetospirillum magneticum AMB 1 x uid58527 223Marinobacter aquaeolei VT8 uid59419 x 224 Methanobrevibacter smithiiATCC 35061 x uid58827 225 Methanoculleus bourgensis MS2 uid171377 x 226Methanolobus psychrophilus R15 x uid177925 227 Methanomethylovoranshollandica DSM x 15978 uid184864 228 Methanosarcina acetivorans C2Auid57879 x 229 Methanosarcina mazei Go1 uid57893 x 230 Methylophagaaminisulfidivorans MP x 231 Microcystis aeruginosa PCC 9443 x 232Nitratireductor indicus C115 x 233 Nostoc punctiforme PCC 73102 uid57767x 234 Opitutacea x 235 Oribacterium sp. ACB1 x 236 Oribacterium sp. ACB7x 237 Oribacterium sp. ACB8 x 238 Paenibacillus elgii B69 x 239 Pantoeasp. GM01 x 240 Parabacteroides sp. D25 x 241 Parvularcula bermudensisHTCC2503 x uid51641 242 Pectobacterium carotovorum PCC21 x uid174335 243Pectobacterium wasabiae CFBP 3304 x 244 Pelobacter propionicus DSM 2379uid58255 x 245 Pelodictyon phaeoclathratiforme BU 1 x uid58173 246Photobacterium sp. SKA34 x 247 Photorhabdus asymbiotica uid59243 x 248Polaromonas JS666 uid58207 x 249 Proteus mirabilis WGLW6 x 250Pseudoalteromona x 251 Pseudoalteromonas luteoviolacea B = x ATCC 29581252 Pseudomonas brassicacearum NFM421 x uid66303 253 Pseudomonas fragiA22 x 254 Pseudomonas fuscovaginae UPB0736 x 255 Pseudomonas mandeliiJR-1 x 256 Pseudomonas psychrotolerans L19 x 257 Pseudomonas stutzeriCCUG 29243 x uid168379 258 Pseudomonas syringae Lz4W x 259 Psychrobactercryohalolentis K5 uid58373 x 260 Rhodobacter sphaeroides ATCC 17025 xuid58451 261 Rhodococcus erythropolis PR4 uid59019 x 262Rhodopseudomonas palustris TIE 1 x uid58995 263 Roseburia inulinivoransDSM 16841 x 264 Roseobacter sp. MED193 x 265 Ruminococcaceae bacteriumD16 x 266 Runella slithyformis DSM 19594 uid68317 x 267 Saccharophagusdegradans 2 40 uid57921 x 268 Salmonella enterica serovar Typhimurium x14028S uid86059 269 Salmonella enterica serovar Typhimurium x 798uid158047 270 Salmonella enterica serovar Typhimurium x LT2 uid57799 271Salmonella enterica serovar Typhimurium x SL1344 uid86645 272 Salmonellaenterica serovar Typhimurium x ST1660 06 uid190371 273 Salmonellaenterica serovar Typhimurium x ST4 74 uid84393 274 Salmonella entericaserovar Typhimurium x STm1 uid181283 275 Salmonella enterica serovarTyphimurium x STm12 uid181362 276 Salmonella enterica serovarTyphimurium x STm2 uid181284 277 Salmonella enterica serovar Typhimuriumx STm3 uid181357 278 Salmonella enterica serovar Typhimurium x STm4uid181358 279 Salmonella enterica serovar Typhimurium x STm8 uid181355280 Salmonella enterica serovar Typhimurium x STm9 uid181356 281Salmonella enterica serovar Typhimurium x T000240 uid84397 282Salmonella enterica serovar Typhimurium x uid86061 283 Salmonellaenterica serovar Typhimurium x UK 1 uid87049 284 Salmonella entericasubsp. enterica serovar x 4,[5],12:i:- str. CVM23701 285 Salmonellaenterica subsp. enterica serovar x Enteritidi 286 Salmonella entericasubsp. enterica serovar x Infanti 287 Salmonella enterica subsp.enterica serovar x Saintpau 288 Salmonella enterica subsp. entericaserovar x Typhimuriu 289 Salmonella enterica subsp. enterica serovar xUganda str 290 Selenomonas sputigena ATCC 35185 x 291 Selenomonassputigena ATCC 35185 x uid55329 292 Shewanella ANA 3 uid58347 x 293Shewanella baltica OS625 x 294 Shewanella MR 4 uid58345 x 295Sinorhizobium meliloti CCNWSX0020 x 296 Slackia heliotrinireducens DSM20476 x uid59051 297 Sphingobium indicum B90A x 298 Sphingobiumyanoikuyae XLDN2-5 x 299 Spirosoma linguale DSM 74 uid43413 x 300Sporolactobacillus vineae DSM 21990 = x SL153 301 Sporosarcinanewyorkensis 2681 x 302 Streptomyces sp. SPB78 x 303 Sulfuricurvumkujiense DSM 16994 x uid60789 304 Synechococcus PCC 6312 uid182934 x 305Syntrophus aciditrophicus SB uid58539 x 306 Thauera MZ1T uid58987 x 307Thermacetogenium phaeum DSM 12270 x uid177811 308 Thermoanaerobacteriumx thermosaccharolyticum DSM 571 uid51639 309 Thiocystis violascens DSM198 uid74025 x 310 Thioflavicoccus mobilis 8321 uid184343 x 311Thiothrix nivea DSM 5205 x 312 Vibrio cholera CIRS 101 x 313 Vibriocholerae 4260B x 314 Vibrio cholerae CP1035(8) x 315 Vibrio choleraeCP1048(21) x 316 Vibrio cholerae CP1050(23) x 317 Vibrio cholerae H1 x318 Vibrio cholerae HC-17A1 x 319 Vibrio cholerae HC-17A2 x 320 Vibriocholerae HC-19A1 x 321 Vibrio cholerae HC-22A1 x 322 Vibrio choleraeHC-23A1 x 323 Vibrio cholerae HC-28A1 x 324 Vibrio cholerae HC-32A1 x325 Vibrio cholerae HC-37A1 x 326 Vibrio cholerae HC-38A1 x 327 Vibriocholerae HC-39A1 x 328 Vibrio cholerae HC-40A1 x 329 Vibrio choleraeHC-41A1 x 330 Vibrio cholerae HC-43A1 x 331 Vibrio cholerae HC-46A1 x332 Vibrio cholerae HC-47A1 x 333 Vibrio cholerae HC-48A1 x 334 Vibriocholerae HC-48B2 x 335 Vibrio cholerae HC-49A2 x 336 Vibrio choleraeHC-55B2 x 337 Vibrio cholerae HC-56A2 x 338 Vibrio cholerae HC-57A2 x339 Vibrio cholerae HC-60A1 x 340 Vibrio cholerae HC-61A1 x 341 Vibriocholerae HC-61A2 x 342 Vibrio cholerae HC-62A1 x 343 Vibrio choleraeHC-62B1 x 344 Vibrio cholerae HC-64A1 x 345 Vibrio cholerae HC-65A1 x346 Vibrio cholerae HC-67A1 x 347 Vibrio cholerae HC-68A1 x 348 Vibriocholerae HC-69A1 x 349 Vibrio cholerae HC-70A1 x 350 Vibrio choleraeHC-71A1 x 351 Vibrio cholerae HC-72A2 x 352 Vibrio cholerae HC-77A1 x353 Vibrio cholerae HC-7A1 x 354 Vibrio cholerae HC-80A1 x 355 Vibriocholerae HC-81A1 x 356 Vibrio cholerae HC-81A2 x 357 Vibrio choleraeHCUF01 x 358 Vibrio cholerae HFU-02 x 359 Vibrio cholerae MJ 1236uid59387 x 360 Vibrio cholerae MO10 x 361 Vibrio cholerae O1 2010EL 1786uid78933 x 362 Vibrio cholerae O1 str. 2010EL-1792 x 363 Vibrio choleraeO1 str. 2010EL-1798 x 364 Vibrio cholerae O1 str. EC-0009 x 365 Vibriocholerae O1 str. EC-0012 x 366 Vibrio cholerae O1 str. EC-0027 x 367Vibrio cholerae O1 str. EDC-020 x 368 Vibrio cholerae O1 str. EM-1546 x369 Vibrio cholerae O1 str. Inaba G4222 x 370 Vibrio cholerae O1 str.Nep-21106 x 371 Vibrio cholerae O1 str. Nep-21113 x 372 Vibrio choleraeO1 str. NHCC-004A x 373 Vibrio cholerae O1 str. NHCC-006C x 374 Vibriocholerae O1 str. NHCC-010F x 375 Vibrio cholerae O1 str. PCS-023 x 376Vibrio cholerae VC4370 x 377 Vibrio harveyi CAIM 1792 x 378 Vibrioshilonii AK1 x 379 Vibrio tubiashii ATCC 19109 x 380 Vibrio choleraeHC-20A2 x 381 Vibrio cholerae HC-21A1 x 382 Vibrio cholerae HC-42A1 x383 Vibrio cholerae HC-51A1 x 384 Vibrio cholerae O1 str. 3582-05 x 385Xanthomonas vesicatoria ATCC 35937 x 386 Yersinia ruckeri ATCC 29473 x387 Zymomonas mobilis NCIMB 11163 x uid41019 388 Clostridium clariflavumDSM 19732 xx Genome contains two BREX uid82345 systems of type #1 389Clostridium saccharolyticum WM1 xx Genome contains two BREX uid51419systems of type #1 390 Fusobacterium necrophorum subsp xx Genomecontains two BREX systems of type #1 391 Gallionella capsiferriformansES 2 uid51505 xx Genome contains two BREX systems of type #1 392Magnetospirillum sp. SO-1 xx Genome contains two BREX systems of type #1393 Methanoplanu xx Genome contains two BREX systems of type #1 394Methanospirillum hungatei JF 1 uid58181 xx Genome contains two BREXsystems of type #1 395 Salmonella enterica serovar Typhimurium xx Genomecontains two BREX STm11 uid181361 systems of type #1 396 Stomatobaculumlongum (Lachnospiraceae xx Genome contains two BREX bacterium ACC2)systems of type #1 397 Syntrophomonas wolfei Goettingen xx Genomecontains two BREX uid58179 systems of type #1 398 Vibrio cholerae B33 xxGenome contains two BREX systems of type #1 399 Haloarcula argentinensisDSM 12282 x 400 Haloarcula hispanica ATCC 33960 x uid72475 401Halobacterium salinarum R1 uid61571 x 402 halophilic archaeon DL31uid72619 x 403 Halopiger xanaduensis SH6 uid68105 x 404 Halorhabdusutahensis DSM 12940 x uid59189 405 Halorubrumlacus profundi ATCC 49239 xuid58807 406 Halosimplex carlsbadense 2-9-1 x 407 Natrinema pellirubrumDSM 15624 x uid74437 408 Natronorubrum tibetense GA33 x 409Anaeromyxobacter dehalogenans 2CP 1 x uid58989 410 Planctomyceslimnophilus DSM 3776 x uid48643 411 Rhodopirellula sp. SWK7 x 412Haliangium ochraceum DSM 14365 xx x Genome contains two BREX uid41425systems of type #6 and one ot type #2 413 Nitrococcus mobilis Nb-231 x xGenome contains two BREX systems: #3 and #2 414 Anaerobaculum mobile DSM13181 x x Genome contains two BREX uid168323 systems: #3 and #4 415Acetohalobium arabaticum DSM 5501 x uid51423 416 Acidothermuscellulolyticus 11B uid58501 x 417 Alcanivorax hongdengensis A-11-3 x 418Bacillus cereus 03BB108 x 419 Bacillus cereus BAG2X1-1 x 420 Bacillusmethanolicus MGA3 x 421 Bacillus cereus W x 422 Bacteroides vulgatusATCC 8482 uid58253 x 423 Caldicellulosiruptor kristjanssonii 177R1B xuid60393 424 Caloramator australicus RC3]Length = 15 x 425 Chloroflexusaggregans DSM 9485 x uid58621 426 Clostridium papyrosolvens DSM 2782 x427 Clostridium thermocellum YS x 428 Desulfotomaculum nigrificans DSM574 x 429 Desulfovibrio aespoeensis Aspo 2 uid42613 x 430Dethiosulfovibrio peptidovorans DSM x 11002 431 Dichelobacter nodosusVCS1703A x uid57643 432 Ectothiorhodospir x 433 Enterococcus faecium1,231,501 x 434 Kingella denitrificans ATCC 33394 x 435 Lachnospiraceaebacteriu x 436 Methanocaldococcus FS406 22 uid42499 x 437 Methanosalsumzhilinae DSM 4017 x uid68249 438 Methylacidiphilum infernorum V4uid59161 x 439 Methyloversatilis universalis FAM5 x 440 Nitrosococcusoceani AFC27 x 441 Nitrosococcus oceani ATCC 19707 x uid58403 442Nitrosococcus watsonii C 113 uid50331 x 443 Pelotomaculumthermopropionicum SI x uid58877 444 Planctomyces brasiliensis DSM 5305 xuid60583 445 planctomycete KSU-1 x 446 Pseudoalteromonas marina mano4 x447 Pseudomonas sp. GM55 x 448 Syntrophothermus lipocalidus DSM 12680 xuid49527 449 Thermanaerovibrio acidaminovorans DSM x 6589 uid41925 450Thermoanaerobacter brockii finnii Ako 1 x uid55639 451Thermoanaerobacter ethanolicus CCSD1 x 452 Thermoanaerobacterethanolicus JW 200 x 453 Thermoanaerobacter italicus Ab9 uid46241 x 454Thermoanaerobacter pseudethanolicus x ATCC 33223 uid58339 455Thermoanaerobacterium saccharolyticum x JW SL YS485 uid167781 456Thermoanaerobacterium x thermosaccharolyticum M0795 uid184821 457Thermoanaerobacterium xylanolyticum LX x 11 uid63163 458Thermoplasmatales archaeon SCGC AB- x 539-N05 459 Treponema primitiaZAS-1 x 460 Vibrio scophthalmi LMG 19158 x 461 Parvibaculumlavamentivorans DS 1 xx Genome contains two BREX uid58739 systems oftype #3 462 Amycolatopsis azurea DSM 43854 x 463 Bradyrhizobium sp. ORS375 x 464 Burkholderia thailandensis E264 x 465 Burkholderiathailandensis E264 uid58081 x 466 Candidatus Accumulibacter phosphatis xclade IIA UW 1 uid59207 467 Corallococcus coralloides DSM 2259 xuid157997 468 Corynebacterium variabile DSM 44702 x uid62003 469 Dietziacinnamea P4 x 470 Frankia CcI3 uid58397 x 471 Frankia EuI1c uid42615 x472 Frankia sp. EUN1f x 473 Gemmata obscuriglobus UQM 2246 x 474Gordonia amicalis NBRC 100051 = JCM x 11271 475 Gordoniapolyisoprenivorans NBRC 16320 x 476 Hahella chejuensis KCTC 2396uid58483 x 477 Micromonospora aurantiaca ATCC 27029 x uid42501 478Micromonospora sp. ATCC 39149 x 479 Mycobacterium gilvum PYR GCK xuid59421 480 Mycobacterium xenopi RIVM700367 x 481 Mycobacteriumintracellulare ATCC 13950 x 482 Nocardia cyriacigeorgica GUH 2 uid89395x 483 Phaeospirillum molischianum DSM 120 x 484 Planctomyces maris DSM8797 x 485 Polaromonas naphthalenivorans CJ2 x uid58273 486 Pseudomonasstutzeri NF13 x 487 Rhodococcus triatomae BKS 15-14 x 488 Rhodococcusruber BKS 20-38 x 489 Saccharomonospora cyanea NA-134 x 490Saccharomonospora glauca K62 x 491 Saccharomonospora viridis DSM 43017 xuid59055 492 Saccharomonospora xinjiangensis XJ-54 x 493Saccharopolyspora erythraea NRRL 2338 x 494 Saccharopolyspora erythraeaNRRL 2338 x uid62947 495 Saccharothrix espanaensis DSM 44229 x uid184826496 Singulisphaera acidiphila DSM 18658 x uid81777 497 Sorangiumcellulosum So ce 56 uid61629 x 498 Streptomyces coelicolor A3 2 uid57801x 499 Streptomyces griseus NBRC 13350 x uid58983 500 Streptomycesturgidiscabies Car8 x 501 Streptomyces gancidicus BKS 13-15 x 502Thermobifida fusca YX uid57703 x 503 Thermobispora bispora DSM 43833 xuid48999 504 Aciduliprofundum MAR08 339 uid184407 x 505 CandidatusDesulforudis audaxviator x MP104C uid59067 506 Coprothermobacterproteolyticus DSM 5265 x uid59253 507 Cyanobacterium stanieri PCC 7202 xuid183337 508 Denitrovibrio acetiphilus DSM 12809 x uid46657 509Desulfitobacterium dichloroeliminans LMG x P 21439 uid82555 510Geobacter M21 uid59037 x 511 Prevotella denticola F0289 uid65091 x 512Thermomicrobium roseum DSM 5159 x uid59341 513 Thermotoga petrophila RKU1 uid58655 x 514 Thioalkalivibrio sp. K90mix x

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”,and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

When reference is made to particular sequence listings, such referenceis to be understood to also encompass sequences that substantiallycorrespond to its complementary sequence as including minor sequencevariations, resulting from, e.g., sequencing errors, cloning errors, orother alterations resulting in base substitution, base deletion or baseaddition, provided that the frequency of such variations is less than 1in 50 nucleotides, alternatively, less than 1 in 100 nucleotides,alternatively, less than 1 in 200 nucleotides, alternatively, less than1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides,alternatively, less than 1 in 5,000 nucleotides, alternatively, lessthan 1 in 10,000 nucleotides.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion. Generally, the nomenclature used herein and thelaboratory procedures utilized in the present invention includemolecular, biochemical, microbiological and recombinant DNA techniques.Such techniques are thoroughly explained in the literature. See, forexample, “Molecular Cloning: A laboratory Manual” Sambrook et al.,(1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel,R. M., ed. (1994); Ausubel et al., “Current Protocols in MolecularBiology”. John Wiley and Sons, Baltimore, Md. (1989); Perbal, “APractical Guide to Molecular Cloning”, John Wiley & Sons, New York(1988); Watson et al., “Recombinant DNA”, Scientific American Books, NewYork; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”,Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998);methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202;4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A LaboratoryHandbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of AnimalCells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N. Y.(1994), Third Edition; “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames. B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames. B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Materials and Methods

Genomic Data and Molecular Phylogeny of the pglZ Protein—

A set of 1447 completely sequenced prokaryotic genomes (1336 bacterialand 111 archaeal genomes) were downloaded from the NCBI FTP site(ftp://ftp.ncbi.nih.gov/genomes/Bacteria/) and used for subsequentanalyses. Several pglZ protein sequences were used as a query in aPSI-BLAST search against the 1447 prokaryotic genomes with an inclusionthreshold e-value of 0.001. Proteins that did not contain the pglZdomain or that were <600 amino acids length were filtered out. Theremaining protein sequences were used to build a pglZ tree as follows:Amino acid sequences were aligned using the MAFFT algorithm [Katoh etal. Nucleic acids research (2002) 30: 3059-3066]. The Fourier transformapproximation was disabled, and substitution rates were modeled with JTT[Jones et al. Computer applications in the biosciences: CABIOS (1992) 8:275-282] and BLOSUM45 matrix, which is suitable for diverged sequences.The gene tree was reconstructed using the probabilistic RA×ML algorithm,with 100 bootstrap replicates, substitutions modeled with JTT (Jones etal. 1992), while allowing for rate variability among sites. Forsimplicity, the tree presented in FIG. 1A shows only the 115 pglZprotein sequences that are part of a complete Bacteriophage Exclusion(BREX) system. The brxC/pglY phylogeny tree was built in the samemanner.

Identification of Bacteriophage Exclusion (BREX) Types 1-6

System types were characterized based on manual observation of phyleticclusters in the pglZ tree. The specific genes associated with each pglZphyletic type were defined using the IMG genome browser(www://img(dot)jgi(dot)doe(dot)gov/cgi-bin/w/main(dot)cgi). Arepresentative protein sequence of each of the individual genes (Table 1below) was then used as query in a PSI-BLAST search with an inclusionthreshold e-value of 0.05. Only gene clusters containing the two coregenes (pglz and brxC/pglY) and at least two additional genes wereconsidered, under the added constraint that the genomic distance betweenthe first and last genes in the system be under 30 kb. In the case ofpglY, homology was based on the shared motifs (the p-loop motifGXXXXGK(T/S) (DUF2791, SEQ ID NO: 6162) and DUF499 combined with theconserved size of the gene in the different subtypes (1200 amino acids).The filtered clusters were manually assigned to systems according togene content. Only clusters containing the complete set of genes ormissing one non-core gene were included in the final set (Tables 2-7below). In the case of BREX type 2, systems missing both brxD and brxHIwere also included in the final set. The blastx program was used to scanintergenic regions in the clusters for unnanotated genes. Proteindomains were annotated using the conserved domain database (CDD)³⁴ andHHpred³⁵. In the latter case, queries were carried out usingrepresentative sequences against the PDB, SCOP, interpro, pfam, smart,tigrfam and COG databases using default search parameters. The blastxprogram was used to scan intergenic regions in the clusters forun-annotated genes.

The consensus organisms tree (represented in FIG. 1A-B) was derived fromthe NCBI “common tree” downloaded from the NCBI Taxonomy portal. Inorder to check whether subtypes were overrepresented in specificbacterial phyla the two following ratios were compared, using ahypergeometric statistical test: (i) Number of instances of a specificsubtype in a specific phylum/total number of the specific subtype inbacteria; (ii) Total number of genomes of the specific phylumanalyzed/total number of bacterial genomes analyzed. P-value ≤0.05 wasconsidered statistically significant following Benjamini and Hochbergcorrection for multiple testing.

Extensive Identification of BREX Systems in Prokaryotic Genomes

A set of 2263 completely sequenced prokaryotic genomes and 5493 draftgenomes was downloaded from the NCBI FTP site(ftp://ftp.ncbi.nih.gov/genomes/Bacterial andftp://ftp.ncbi.nih.gov/genomes/Bacteria_DRAFT/, respectively) and usedfor subsequent analyses. A representative protein sequence of each ofthe 13 genes (Table 1 below) was then used as query in a PSI-BLASTsearch against the 7756 completely sequences and draft genomes with aninclusion threshold e-value of 0.05. Only gene clusters containing thetwo core genes (pglZ and brxC/pglY) and at least two additional geneswere considered and listed in tables 10-15 below.

Strain Construction—

The type 1 BREX system was amplified in fragments from the Bacilluscereus H3081.97 genome from position 89,288-103,514 (GenBankABDL02000007.1, SEQ ID NO: 6164). The PCR-amplified fragments wereassembled to a circular plasmid in S. cerevisiae using the pYES1L vector(Invitrogen), transformed into Eccherichia coli BL21 AI and amplified,and then integrated into the proB gene in Bacillus subtilis BEST7003,along with a chloramphenicol resistance cassette. The DNA sequence ofthe plasmid used for the integration is depicted in SEQ ID NO: 6139. Theprimers used for construction are depicted in SEQ ID NO: 6140-6151. Thepresence of the intact BREX system within Basillus subtilis BEST7003 wasconfirmed by PCR and Illumina-based whole genome sequencing. Primerssequences are depicted in SEQ ID NO: 6152-6161. Control strains containonly the chloramphenicol resistance cassette integrated at the proBlocus. The pglX deletion strain was constructed in a similar manner withPCR fragments that created a deletion from position 94,655-98,163(GenBank ABDLO2000007.1, SEQ ID NO: 6164), leaving only 31 nucleotidesof the pglX gene. The DNA sequence of the plasmid used for theintegration is depicted in SEQ ID NO: 6210.

Growth Dynamics of Phage Infected Cultures—

Overnight cultures were diluted 1:100 in LB media supplemented with 0.1mM MnCl₂ and 5 mM MgCl₂ and then grown to an OD₆₀₀ of 0.06 in a 96-wellplate format. Phages were added at a multiplicity of infection (MOI)ranging from 10⁻³ to 10⁻⁴. High concentration phage infections wereperformed at MOI ranging from 0.05 to 5. Optical density measurements ata wavelength of 600 nm were taken every 13 minutes using a TECANinfinite 2000 plate reader.

Plaque Assays—

Small drop plaque assays were initially performed using 0.75% agarplates containing bacterial cultures that were diluted 1:13 in LB mediasupplemented with 0.1 mM MnCl2 and 5 mM MgCl₂. Serial dilutions of thephage between 2×10⁰ and 2×10⁵ plaque-forming units (pfu) were spotted onthese plates and plaques were counted after overnight growth at roomtemperature. Further confirmation of plaque numbers was performed by anagar overlay assay. The bottom agar was composed of LB mediasupplemented with 0.1 mM MnCl₂ and 5 mM MgCl₂ and 1.5% agar. The topagar was prepared by diluting overnight bacterial cultures 1:30 in LBmedia supplemented with 0.1 mM MnCl₂ and 5 mM MgCl₂ and 0.5% agar withthe addition of serial dilutions of the phage. Plaques were countedafter overnight growth at room temperature.

One-Step Phage Growth Curve Assays—

One-step phage growth curve experiments were performed as described byCarlson [E. Kutter and A. Sulakvelidze (ed.), Bacteriophages: biologyand applications, CRC Press, Boca Raton. Fla. Appendix p. 437-494].Logarithmic phase cultures were infected with either phage SPO1 or ϕ3Tat an MOI of 0.05. Following 18 minutes of growth at 37° C. the infectedculture was diluted 1:10,000, to reduce the likelihood of phageinfection following cell lysis. To evaluate the number of infectivecenters and extracellular phage present in the infected culture, sampleswere taken at specific time points throughout the incubation period,mixed with a phage-sensitive Basillus subtilis strain and plated usingthe agar overlay method described hereinabove. Phage adsorption wasinferred by evaluating the number of extracellular phage present in themixture 15 minutes following infection. This was assayed by mixing theinfection mixture with chloroform, incubating it at 37° C. for 4minutes, followed by incubation for 4 minutes on ice, and 30 minutes atroom temperature. The aqueous phase was then mixed with aphage-sensitive Basillus subtilis strain and plated using the agaroverlay method described hereinabove. The addition of chloroform leadsto bacteria killing, including phage adsorbed bacteria. At early timepoints, phage have not yet assembled inside the cell, and are thereforeunable to form plaques. Thus, the derived results allow evaluation ofthe extracellular phage levels. A drop in extracellular phage levelsindicates that adsorption has occurred.

DNA Extraction—

DNA extraction was performed by suspending cells in 50 mM EDTA pH 8.0with a lytic enzyme (lysozyme. Sigma) for 90 minutes at 37° C. followedby centrifugation for 2 minutes at 13,000 g and removal of supernantat.The cells were then lysed by adding a Nuclei Lysis Solution (Promega,cat no. A7941) for 5 minutes at 80° C. followed by addition of Rnase A(10 mg/ml) for 30-60 minutes at 37° C. The protein fraction wasprecipitated by adding 200 μl Protein Precipitation Solution (Promega,cat no. A795A), incubating the suspension for 5 minutes on ice followedby centrifugation at 13,000-16,000 g for 3 minutes. The supernatant wasthen transferred to a clean tube containing 600 μl isopropanol, mixedand centrifuged at 13,000-16,000 g for 3 minutes. The supernatant wasremoved and 600 μl 70% Ethanol was added to the pellet mixed andcentrifuged at 13,000-16,000 g for 3 minutes. The ethanol was thenaspirated and the pellet was air dried for a couple of minutes followedby resuspension in Qiagen Elution buffer.

Phage Infection Time Courses, Genomic DNA Sequencing and MethylationAnalysis—

Phage infection time course cultures for both methylome analysis,detection of lysogeny and relative phage abundance were performed at anMOI of 4. Phage infection time course cultures are practically culturesinfected by phage and analyzed at specific time points (e.g. 0, 5, 10,15, 20, 30 and 40 minutes following infection). Uninfected culturesanalyzed at the same time points served as control. Cell pellets werewashed three times in 10 mM Tris pH 7.4 to remove unadsorbed phage,followed by DNA extraction as described hereinabove. DNA librarypreparations and sequencing for methylome analysis were performed at theYale Center for Genome Analysis (see Murray I A et al. (2012) Nucleicacids research 40: 11450-11462). To determine the relative abundance ofbacterial and phage ϕ3T DNA levels, DNA was first fragmented usingNEBNext® dsDNA Fragmentase (New England Biolabs Ibc.) according tomanufacturer's instructions, followed by Illumina sequencing of the DNAlibraries of ϕ3T phage-infected time course cultures. The sequences weremapped to the phage and host genomes as previously described [Wurtzel etal. PloS one (2010) 5: e15628]. Sequences shared by both Basillussubtilis BEST7003 and phage ϕ3T DNA were discarded from the dataset. Theremaining mapped sequences were enumerated at each time point to comparethe number of sequences mapped to the Basillus subtilis BEST7003 DNArelative to phage ϕ3T DNA and normalized to the genome size.

Detection of Phage Lysogeny—

Genomic DNA sequencing of a lysogen containing phage ϕ3T was performedusing Illumina sequencing to determine the DNA sequence of the ϕ3T phageand the site of phage integration in the genome. The integration of theϕ3T phage was determined at a GTAGG site on the Basillus subtilisBEST7003 bacterial genome at position 2106060-2106064. Multiplex PCRassays were used to detect phage ϕ3T DNA, Basillus subtilis BEST7003DNA, and the novel junction created in the lysogenized strain. Primersused to detect phage ϕ3T were GAGGTTCGCTACGGGCGAAAT (SEQ ID NO: 6211)and TCTCTGCTTGATITCGTCCATGA (SEQ ID NO: 6212). Primers for detection ofBasillus subtilis BEST7003 and the unique junction found in the lysogenwere TGCCTGCATGAGCTGATITG (SEQ ID NO: 6213) and GCAGGAATGAATGGTGGATATTG(SEQ ID NO: 6214); and TCATGCTCCGGATTTGCGAT (SEQ ID NO: 6215) andTGCCTCCITTCGATITTGTTACC (SEQ ID NO: 6216), respectively.

Structural Homology Between brxA and NusB—

Alignment between brxA from Magnetospirillum magneticum (PDB entry 3BHW)and NusB from Aquifex aelicus (PDB entry 3R2C) was performed using theMultiProt web server and presented using PyMol (Schrödinger, Inc,Portland, Oreg., USA).

Agarose Gel and Southern Blot Analysis—

200 ng of undigested genomic DNA was run on a TAE agarose gel. Theagarose gel was depurinated in 0.25 N HCl for 20 minutes, rinsed inddH₂O, and soaked in denaturation buffer (0.5 M NaOH, 1.5 M NaCl) for 10minutes. The DNA was then transferred onto HybondXL membrane (Amersham)by capillary transfer in denaturation buffer and the membranes werebaked for 2 hours at 80° C. DNA for probes was labeled with ∝³²P-dCTPusing the High Prime Kit (Roche Cat no. 11 585 584 001) according tomanufacturer's instructions. Phage ϕ3T specific primers were PTG111:TGGATTTCAGCTGGGGAAGA (SEQ ID NO: 6217) and PTG112:AACTTGTCTCTATCTTATCACCTGT (SEQ ID NO: 6218). The membranes wereincubated overnight with the probe at 65° C. in hybridization buffer (7%SDS, 0.5 M NaPhosphate pH 7.2, 10 mM EDTA), washed twice with 2×SSC,0.1% w/v SDS, washed twice with 1×SSC, 0.1% w/v SDS, then four timeswith 0.2×SSC, 0.1% w/v SDS and exposed to phosphorimager screen andvisualized.

RNA sequencing and 5′ and 3′ RACE—were performed as described in WurtzelO. et al. (2012) Molecular Systems Biology, 8:583.

Example 1 Bacteriophage Exclusion (BREX) System is Abundant in Bacteriaand Archaea

Previous reports demonstrated that various combinations of genesbelonging to the Phage Growth Limitation (PGL) system, and predominantlypglZ, were enriched within ‘defense islands’ of bacteria andarchaea^(9,13). The present inventors have initially performed homologysearches in 1447 bacterial and archaeal genomes in order to understandwhether there is higher order organization amongst pglZ and itsassociated genes. These homology searches found 144 occurrences of pglZamongst the 1447 bacterial and archaeal genomes analyzed. Phylogenetictree reconstruction of these pglZ proteins showed clear clustering ofpglZ into several defined phyletic groups (FIG. 1A). By analyzing thegenomic context of pglZ in each of these groups, a distinct set of 13genes strongly associated with pglZ was identified (Table 1 below). Thecomposition and order of these genes were highly coherent within eachphyletic group but differed between the clades, defining clearorganizational subtypes, with each subtype composed of 4-8 genes. Of the14 genes, only brxC/pglY and pglZ recurred in all system subtypes, withthe additional genes being subtype-specific.

The present inventors termed this overall system as ‘BREX’(Bacteriophage Exclusion, previously termed PYZA), and defined six majorBREX types according to the phylogeny and operon organization (FIG. 1A).Thus, overall 135 BREX systems were found in 9% (126/1447) of allgenomes analyzed, usually appearing on the chromosomal DNA (Tables 2-8above). BREX type 1, the most common form of BREX, appeared 79 times in75 genomes (Table 8 below), and is typically composed of 6 genesarranged in a conserved order (FIG. 1A).

Taken together pan genomic analysis revealed a novel broadly distributedmulti-gene system which the present inventors denoted BREX system. Thisfamily of systems exists in almost 10% of sequenced microbial genomes,and can be divided into six coherent subtypes in which the genecomposition and order is conserved (for further details see Example 2below). Each BREX subtype contains 4-8 genes. By definition, all BREXsubtypes contain a pglZ-domain gene. In addition, all of them harbor alarge protein with a P-loop motif. The P-loop motif (GXXXXGK[T/S]) is aconserved ATP/GTP binding motif that is ubiquitously found in manyATP-utilizing proteins such as kinases, helicases, motor proteins andproteins with multiple other functions [Thomsen and Berger Molecularmicrobiology (2008) 69: 1071-1090]. In general, the P-loop containinggenes in the various BREX subtypes share little homology: for example,the brxC gene of BREX type 1 and pglY gene of BREX type 2 share homologyonly across 4% of their protein sequence, and this homology isconcentrated around the P-loop motif (FIG. 2). Despite the low homology,distant homology analysis with HHpred [Soding Bioinformatics (2005) 21:951-960] showed that they share a domain denoted DUF499 (Table 1). It istherefore suggested that the P-loop containing genes in all six BREXsubtypes share a similar role in the system, and hence these genes aredenoted herein as brxC/pglY and referred to as having a common function(Table 1). Apart from the two core genes pglZ and brxClpglY that appearin each of the six BREX subtypes, the remaining genes aresubtype-specific or restricted to only a subset of the BREX subtypes.

TABLE 1 Genes composing the BREX systems Subsystems in which gene Mediangene Gene appears Associated domains Domain annotation size (aa) pglZCore gene pfam08665 Alkaline phosphatase 835 brxC/ Core gene DUF499, ATPbinding 1208 pglY DUF2791 (pfam10923) brxA 1, 3, 5, 6 DUF1819 Unknownfunction 232 (pfam08849) pglX 1, 2, 5, 6 Pfam13659 Adenine-specific 1175(COG1002/COG0286) methylase brxL 1, 4 COG4930 Lon-like protease 682brxHII 3, 5 COG0553 DNA/RNA helicases 965 brxHI 2, 6 COG1201 Lhr-likeHelicase 712 brxD 2, 6 DUF2791 ATP binding 442 (pfam10923) brxE 6Unknown function 201 brxB 1, 5, 6 DUF1788 Unknown function 193(pfam08747) pglXI 3 COG0863/COG1743 Adenine-specific 920 (pfam01555)methylase brxF 3 ATPase 158 pglW 2 COG0515 Serine/threonine protein 1413kinase brxP 4 COG0175 Phosphoadenosine 774 (pfam01507), phosphosulfate,reductase pfam13182

Example 2 Characterization of the Six BREX Types

Six types of BREX system were characterized based on manual observationof phyletic clusters in the pglZ tree (FIGS. 1A-B and Tables 2-8 above).

Type 1 BREX—

The most common BREX system identified comprises a 6-gene clusterarranged in a highly conserved order in a diverse array of bacteria andarchaea (FIG. 1A-C and Table 2 above). Two of the six genes found inthis conserved cluster share homology with genes from the previouslyreported Pgl system¹¹: pglZ, coding for a protein with a predictedalkaline phosphatase domain, and pglX, coding for a protein with aputative methylase domain. The four additional genes include (i) aIon-like protease-domain gene, denoted herein as brxL; (ii) a gene,denoted herein as brxA; (iii) a gene, denoted herein as brxB; and (iv) alarge, ˜1200 amino acid protein with an ATP binding motif(GXXXXGK[T/S]), denoted herein as brxC. Although this does not resembleany classical combination of genes currently known to be involved inphage defense, the preferential localization of this conserved genecluster in the genomic vicinity of other defense genes suggests that itcould form a novel phage defense system.

The brxA family of proteins are, on average, 232 amino acids long and donot share sequence similarity with any domain of known function.However, as part of the protein structure initiative the structure ofthe type 1 brxA protein from Magnetospirillum sp. SO-1 was solved (PDBentry 3BHW). A significant structural similarity, spanning 44 aminoacids of the brxA protein, was found between the Magnetospirillum brxAand the 148 amino acids RNA binding protein NusB (PDB entry 3R2C)[Stagnoet al. Nucleic acids research (2011) 39, 7803-7815]. NusB is part of ananti-termination complex that enables proper ribosomal RNA transcriptionin E. coli. The anti-termination complex is initiated by binding of NusBand NusE to a BOXA site, a specific sequence on the nascent rRNA. Thecomplex, which assembles additional proteins such as NusE, NusG andNusA, modifies RNA polymerase to enable readthrough past Rho-dependenttranscriptional terminators that are present in the rRNA sequence[Luttgen et al. Journal of molecular biology (2002) 316, 875-885]. NusBwas also shown to be essential for the life cycle of bacteriophage λ,and specifically for the transition from early transcription into latetranscription. In the middle stages of infection, the phage N proteincouples with NusB. NusE, NusA and NusG to direct the host RNA polymeraseto read through the terminators of the phage immediate early genes andproceed to transcription of middle genes [Stagno et al. Nucleic acidsresearch (2011) 39, 7803-7815]. As demonstrated in FIG. 3, the brxAprotein displays significant structural homology to NusB. Thissimilarity spans the RNA-binding interface, as well as part of theprotein:protein interaction interface with NusE. In light of thissimilarity, it is proposed that brxA is also an RNA binding protein. Itis further speculated that this protein has a role in interfering withthe phage infection cycle by disrupting anti-termination eventsessential for the phage cycle.

Type 2 BREX—

Type 2 BREX system encloses the phage defense system originallydescribed as PGL¹⁰ (FIGS. 1A-B and Table 6 above). However, while thedescribed PGL system composed four genes (pglW, X, Y and Z)¹¹, in 89% ofthe cases (16/18 instances) two additional genes, denoted herein as brxDand brxHI, were found to be associated with the system. Given that boththese genes appear in the same order in the type 6 BREX system (FIG.1A), it is suggested that these genes play an integral part of the type2 system. The first gene, brxD, encodes a small protein predicted tobind ATP, while the second gene, brxHI encodes a predicted helicase. Inaddition, the serine-threonine kinase (pglW) exists exclusively in thissubtype.

Type 3 BREX—

The type 3 BREX system was observed in 20 of the genomes analyzed (FIGS.1A-B and Table 5 above). Both systems type 1 and type 3 contain theshort protein denoted herein as brxA, which has no known function. Inaddition, both type 1 and type 3 systems contain a gene encoding anadenine-specific DNA methylase (pglX and pglXI for subtypes 1 and 3,respectively), although the methylase domain differs between thesubtypes (pfam13659 and pfam01555 in pglX and pglXI, respectively). Itis therefore likely that pglX and pglxXI perform the same DNAmethylation function although they do not share sequence homology. BREXtype 3 systems contain a predicted helicase (denoted herein as brxHII)instead of the ion-like protease present in type 1. In addition, thebrxB gene present in type 1 has been replaced with another protein,denoted herein as brxF.

Type 4 BREX—

The type 4 BREX is composed of four genes (FIGS. 1A-B and Table 7above), two of which are the core brxC/pglY and pglZ genes, and thethird is the lon-like protease (denoted herein as brxL). The fourthgene, denoted herein as brxP, is subtype-specific and contains aphosphoadenylyl-sulfate reductase domain (COG0175/pfam01507). Thisdomain was previously associated with the phage-resistance DND systemthat performs sulfur modifications on the DNA backbone, providing anadditional link between BREX systems and phage resistance¹⁶⁻¹⁸.

Type 5 BREX and Type 6 BREX—

The two least common BREX subtypes, type 5 and type 6, are similar tothe type 1 BREX system but contain some additional variations (FIGS.1A-B and Tables 3 and 4 above). In type 5 BREX, the Ion-like proteasepresent in type 1 BREX has been replaced by a helicase-domain gene(denoted herein as brxHII) carrying a COG0553 domain, and brxC/pglY hasbeen duplicated (FIG. 1A and Table 3 above). In subfamily 6, theprotease present in type 1 has been replaced by two genes, a helicasewith a COG1201 domain (brxHI), and an ATP/GTP binding protein (brxD)(FIG. 1A and Table 4 above); a pair which also appears in type 2 BREX.Type 6 BREX systems also contain an additional gene found as the firstgene in the cluster, which was denoted herein as brxE.

Taken together, 135 of the 144 (94%) pglZ genes detected in microbialgenomes were found to be embedded as part of one of the six BREX systemsdescribed (Table 8 above), and 7 of the remaining pglZ genes wereclearly part of degraded (probably pseudogenized) systems. In most casesa single BREX system per organism was found, with only 8 (6.5%) ofgenomes harboring more than one subtype (Table 8 above). In addition, in14% (19/135) of the identified systems, one of the genes was eithermissing or has become a pseudogene (tables 2-7 above), possiblyrepresenting inactivated systems. A similar tendency for gene loss wasobserved for the CRISPR-Cas system, and it was suggested that CRISPR-Casinactivation is caused by fitness cost imposed by this defensesystem^(2,19,20). Phage defense systems often encode toxic genes²¹, andit is possible that such toxic genes encoded by BREX systems imposefitness cost and lead to gene loss in the absence of phage pressure.

Example 3 Type 1 BREX Confers Resistance to Phage Infection in BacillusSubtilis

To determine whether the BREX system provides protection against phageinfection, the complete type 1 BREX system from Bacillus cereus H3081.97(FIG. 4A), composing the brxA, brxB, brxC, pglX, pglZ, and brxL geneswas integrated into a Bacillus subtilis strain lacking an endogenousBREX system. The type 1 BREX system was integrated into the Bacillussubtilis BEST7003 genome, a derivative of Bacillus subtilis 168 thatlacks the SPβ lysogenic phage, avoiding the potential for superinfectionexclusion. Proper integration of the intact system was verified by PCRand complete genome sequencing. RNA-sequencing further verified that thegenes of the integrated system are transcribed in Bacillus subtilis whengrown in exponential phase in rich medium. Furthermore, using 5′ and 3′RACE it was determined that the system is transcribed as two operonswith the first four genes, brxA-brxB-brxC-pglX, forming a singletranscriptional unit, while the last two genes, pglZ-brxL, areco-expressed as a second transcriptional unit (FIG. 4B). The observationthat the genes in the putative BREX system are co-transcribed as twolong polycistronic mRNAs further supports they work together ascomponents of a functional system.

Ten Bacillus subtilis phages were selected for phage infectionexperiments, spanning a wide range of phage phylogeny, from T4-likeMyoviridae (SPO1 and SP82G), lambda-like Siphoviridae (ϕ105, rho10,rho14 and SPO2) and SPβ-like Siphoviridae (Φ3T, SPβ, SP16 and Zeta). Twoof the phages are obligatory lytic (SPO1 and SP82G), while the remainingare temperate (See Table 9 below). The sensitivity of Bacillus subtilisstrains either lacking or containing the BREX type 1 system to infectionby the different phages was evaluated using both optical densitymeasurements in a 96-well plate format, and double agar overlay andplaque assays (Table 9 below).

Upon phage infection, the Bacillus subtilis strain containing the BREXsystem showed complete resistance to five of the eight temperate phagestested (FIGS. 4D, I-L and Table 9 below). Growth curves ofBREX-containing bacteria infected with these phages wereindistinguishable from the uninfected bacteria, while rapid declines inoptical density measurements were observed for the control strainlacking the BREX system, indicating lysis of the infected cells (FIGS.4D, I-L). These results confirm that BREX is a phage defense system thatprovides protection against a wide array of phages, both virulent andtemperate ones. In contrast, phage resistance was not observed uponinfection with phage Φ105 and its close relatives, rho10 and rho14.Similar kinetics of cell lysis was observed for strains eithercontaining or lacking the BREX system (FIGS. 4E, 4H and 4M and Table 9below). Considering that phage Φ105 is estimated to share high (83-97%)genome homology with rho10 and rho14¹⁴, the inability of the type 1 BREXsystem to protect against these three phages could indicate that thisphage family has evolved strategies to counteract the BREX defense, ashas been observed with other bacterial defense systems¹⁵.

To further evaluate the level of protection provided by the type 1 BREXsystem against the tested temperate phages, plaque assays usingincreasing dilutions of phage were performed. For five of the temperatephages, no plaques were observed when the type 1 BREX-containing strainwas challenged even with the highest phage concentrations, indicatingthat the type 1BREX system provides at least a 10⁵ fold protectionagainst cell lysis upon phage infection (Table 9 below). The plaqueassays also confirmed that phage Φ105 and its relatives evade type 1BREX defense, with similar efficiencies of plating and plaque morphologyobserved in both type 1 BREX-containing and wild-type control strains(Table 9 below).

TABLE 9 Type 1 BREX protection against phage infection InfectionEfficiency blocked of by BREX Phage Genus Family Life cycle BREX?protection^(a) SPβ SPβ-like Siphoviridae Temperate Yes >10⁵ SP16SPβ-like Siphoviridae Temperate Yes >10⁵ Zeta SPβ-like SiphoviridaeTemperate Yes >10⁵ Φ3T SPβ-like Siphoviridae Temperate Yes >10⁵ SPO2Lambda- Siphoviridae Temperate Yes >10⁵ like Φ105 Lambda- SiphoviridaeTemperate No 1 like rho10 Lambda- Siphoviridae Temperate No 1 like rho14Lambda- Siphoviridae Temperate No 1 like SPO1 SPO1-like MyoviridaeObligatory Yes 8 * 10² ± lytic 0.02 SP82G SPO1-like MyoviridaeObligatory Yes 1.8 * 10¹ ± lytic 0.08 ^(a)Protection efficiency wascalculated as the ratio between the number of plaques formed on theBREX-lacking strain divided by the number of plaques formed on theBREX-containing strain with the same phage titer, using increasingtiters. Standard deviation was calculated from a biological triplicateof the plaque experiment.

The type 1 BREX-containing Bacillus subtilis strain also displayed someprotection from the lytic SPO1 and SP82G phages in liquid cultureexperiments. Growth curves of the strain containing the BREX type 1system infected with either SPO1 or SP28G phages were similar to theuninfected strains when evaluated for up to 12 hours followinginfection, while complete lysis was observed in infected control strainslacking the BREX system (FIGS. 4F-G). Endpoint analysis using plaqueassays revealed a 10¹ fold reduction in plaque numbers in type 1BREX-containing strains for SPO1 and SP82G phages (Table 9 above). Inaddition, plaque sizes were reduced 1.5-2 fold in the type 1BREX-containing strain, to 47% and 65% the diameter of those observed inthe control strain lacking the type 1 BREX system for SPO1 and SP82G,respectively (data not shown). These results are consistent with theobservation that incubation of the type 1 BREX-containing strain withthe two lytic phages for extended periods of time (>20 hours) oftenresulted in an eventual culture decline occurring at apparentlystochastic points in time (FIGS. 5A-B).

To gain further insight into the nature of the incomplete type 1 BREXdefense against these lytic phages, a one-step phage growth curve assay[Carlson Bacteriophages, Biology and Applications (eds. E Kutter, ASulakvelidze) (2005) pp. 437-494. CRC Press, Florida.] was performedwith SPO1. Briefly, this experiment involves mixing SPO1-infected cellswith a SPO1-sensitive B. subtilis cells and plating them together usingan agar overlay method. Phage bursts from successful infections arevisualized as a single plaque on a lawn from the SPO1-sensitive B.subtilis strain, enabling an evaluation of the number of phages thathave adsorbed and completed a successful infection cycle. Asdemonstrated in FIG. 6, enumeration of plaques during the first 45minutes of the time course infection indicated that the SPO1 phage wasable to complete the lytic cycle only in 9%±4 of the initially infectedcells (FIG. 6). A delay in kinetics of the phage cycle was alsoobserved, with phage bursts observed 75 minutes and 105 minutesfollowing infection of BREX-lacking and type 1 BREX-containing cells,respectively (FIG. 6).

Taken together, these results suggest that the type 1 BREX systemprovides significant protection from infection by the lytic phages SPO1and SP82G.

Example 4 The Mechanism of Action of Type 1 BREX

Due to the homology of a subset of the genes in the BREX system to genesin the previously described Pgl system¹⁰, it was necessary to examinewhether BREX also functions through the described Pgl mechanism. The Pglphenotype observed in S. coelicolor A3 predicts that the Pgl system doesnot confer resistance to phage first cycle of infection. One-step phagegrowth curve assays were used to examine the first infection cycle ofphage Φ3T in type 1 BREX-containing cells. As demonstrated in FIG. 7,while the control strains lacking the BREX system displayed phage burstsizes of 61.5±10.2 particles per infected cell, there was no productionof Φ3T phage during infections of type 1 BREX-containing Bacillussubtilis strains under similar conditions. To exclude the possibilitythat productive phage infection could occur at later time points,experiments were extended to 120 minutes (corresponds to 3 infectioncycles in control strains) in type 1 BREX-containing Bacillus subtilisstrains. As shown in FIG. 7, plaques were not observed in the type 1BREX-containing Bacillus subtilis strains even at later time points.These results demonstrate that unlike the S. coelicolor Pgl system, thetype 1 BREX system confers resistance to phage first cycle of infection.

Previous experiments with the S. coelicolor Pgl system also demonstratedthat although the Pgl defense system prevents continued propagation ofthe temperate phage ΦC31, it does not block lysogeny of the phage¹⁰. Todetermine whether BREX also permits lysogeny, phage Φ3T integration intothe Bacillus subtilis genome during infection was examined using a PCRassay. In control Bacillus subtilis strains lacking BREX, lysogeny wasfirst detected 10 minutes following phage infection (FIG. 8). However,no evidence for phage integration into the host genome was found in type1 BREX-containing Bacillus subtilis strains. Evaluation of lysogeny inbacterial colonies that survived the phage infection also indicated thatnone of the surviving type 1 BREX-containing colonies were lysogens,while all surviving colonies tested in strains lacking the BREX systemwere lysogenic for phage Φ3T.

One of the common forms of phage defense is abortive infection (Abi),where infected cells commit “suicide” before phage progeny are produced,thus protecting the culture from phage propagation⁴. To test whether thetype 1 BREX system acts via an Abi mechanism, the type 1 BREX-containingBacillus subtilis strains were infected with increasing concentrationsof Φ3T phage. Using high multiplicity of infection (MOI) where nearlyall bacteria are infected in the first cycle, massive cell lysis shouldbe observed in the culture in the case of Abi. The results demonstratedthat even at an MOI>1, no significant growth arrest or culture declinewas found in the liquid culture (FIG. 9), suggesting that the type 1BREX is not an Abi system.

In the next step, BREX ability to prevent phage adsorption and phage DNAreplication were evaluated. As illustrated in FIG. 10, adsorption assaysshowed that Φ3T efficiently adsorbs to both type 1 BREX-containing andBREX-lacking Bacillus subtilis strains, indicating that type 1 BREX doesnot block adsorption. In order to test phage DNA replication withininfected cells, total cellular DNA (including chromosomal DNA andintracellular phage DNA) was extracted at successive time pointsfollowing a high-MOI infection by Φ3T; and the extracted DNA wassequenced by Illumina sequencing. Since host DNA is not degradedfollowing Φ3T infection (FIG. 18), mapping sequenced reads to thereference Bacillus subtilis and Φ3T genomes, allowed quantification ofthe number of Φ3T genome equivalents per infected cell was quantified ateach time point. In control Bacillus subtilis strains lacking BREX,phage DNA replication began between 10 and 15 minutes followinginfection, and 30 minutes following infection, phage DNA levels wereelevated 81-fold relative to that observed at the 10 minutes time point(FIG. 11). In contrast, no increase in phage DNA levels was observed intype 1 BREX-containing Bacillus subtilis strains (FIG. 11).

To further test whether BREX leads to cleavage or degradation of phageDNA, the integrity of phage DNA was examined using Southern blotanalysis on total cellular DNA extracted from phage-infected cells atincreasing time points following infection. This analysis showedextensive replication of phage DNA in control Bacillus subtilis strainslacking type 1 BREX and affirmed no phage DNA replication in Bacillussubtilis strains containing type 1 BREX (FIG. 12). However, as shown inFIG. 12, the phage DNA in type 1 BREX-containing Bacillus subtilisstrains appeared intact with no signs of phage DNA cleavage orprocessive degradation.

These results indicate that phage DNA replication does not occur in type1 BREX-containing cells, that type 1 BREX does not lead to thedegradation of phage DNA and that this system exerts its function at theearly stages of the infection cycle.

As type 1 BREX contain an m6A DNA adenine methylase (pglX), the presentinventors have evaluated whether either bacterial or phage DNA aremethylated in a BREX-dependent manner. To this end, the PacBiosequencing platform that directly detects m6A modifications in sequencedDNA [Murray et al. Nucleic acids research (2012) 40: 11450-11462] wasused. As demonstrated in FIG. 13A, the PacBio platform clearly detectedm6A methylation on the 5^(th) position of the non-palindromic hexamerTAGGAG in chromosomal DNA extracted from type 1 BREX-containing Bacillussubtilis strains. Thus, while nearly all TAGGAG motifs were methylatedin type 1 BREX-containing Bacillus subtilis strains (FIG. 13B), nomethylation on this motif was observed in the control Bacillus subtilisstrains lacking the BREX system. These results indicate that type 1 BREXdrives motif-specific methylation on the genomic DNA of the bacteria inwhich it resides.

To examine whether BREX also methylates the invading phage DNA, totalcellular DNA (including chromosomal DNA and intracellular phage DNA) wasextracted at 10 and 15 minutes following a high-MOI infection by Φ3T andanalysed PacBio sequencing. The results affirmed that the TAGGAG motifsin the bacterial genome were methylated throughout the infection.However, there was no methylation on these motifs in the phage genome atthe time points tested during infection (data not shown).

The presence of bacterial-specific methylation could suggest that thetype 1 BREX system encodes some kind of restriction/modificationactivity, and that the methylation of TAGGAG motifs in the bacterialgenome may serve to differentiate between self and non-self DNA. Thissuggests that deletion of the methylase gene, pglX, would be detrimentalto the cell, as the genomic TAGGAG motifs will no longer be protectedfrom the putative restriction activity of BREX. However, as can be seenin FIG. 14A, deletion of the pglX from the type 1 BREX system that wasintegrated into Bacillus subtilis was not toxic to the cells. Moreover,type 1 BREX-containing Bacillus subtilis strains having a deletion ofpglX were sensitive to all phage tested (for example FIG. 14Bdemonstrating strain sensitivity to Φ3T). These results show that pglXis essential for type 1 BREX-mediated phage resistance, and also suggestthat the BREX mechanism of action is not consistent with a simplerestriction/modification activity.

Taken together, these results suggest that phage adsorption occurs intype 1 BREX-containing strains. This system does not display the Pglphenotype, and hence probably functions through a novel mechanismdifferent than that of the Pgl system. In addition, the systemmethylates the host chromosomal DNA at a specific motif, and that thismethylation is likely to be essential for the system's activity.

Example 5 Extensive Horizontal Transfer of BREX Systems

An examination of the distribution of BREX systems across microbialspecies showed that these systems undergo extensive horizontal transfer(FIG. 15). First, the distribution of systems across the species tree isinterrupted (resembling the way CRISPRs are distributed in bacterial andarchaeal genomes²²). Second, the pglZ tree is not consistent with thespecies tree, and closely related species can accommodate distantlyrelated pglZ and vice versa. Nevertheless, phylogenetic treesreconstructed from additional BREX genes generally recapitulate thestructure of the pglZ tree, suggesting that genes within specific BREXsystems co-evolve and are co-horizontally transferred (For example FIG.16).

The individual clades demonstrated in FIG. 1A separate close to the rootof the PglZ tree and the six defined BREX subtypes are widespread acrossthe entire bacterial and archaeal tree of life (FIG. 15), thussuggesting that the separation between the systems occurred at anancient point in the evolutionary history of bacteria and archaea. Therelative abundance of BREX type 1, and its appearance on several cladeson the pglZ tree (FIG. 1A), suggest that this subtype might be theancestral form of BREX. Despite the extensive horizontal transferobserved for the BREX systems, some clades show enrichment in specificsubtypes: subtype 1 is enriched in Deltaproteobacteria (p=0.001);subtype 2 (the PGL system) appears almost solely in Actinobacteria(p=4.8×10⁻⁹); and subtype 5 is exclusive to the archaeal classHalobacteria. The enrichment of specific subtypes within specific phylamight link the ancestry of these subtypes to the phyla in which they areenriched; alternatively, phylum-specific BREX subtypes might rely onadditional, phylum-specific cellular mechanisms that are not directlyencoded in the BREX genes, or provide defense against phages thatpredominantly attack the specific phyla.

Within the 1447 genomes, the relative frequency of BREX in archaea (10%)was similar to that observed in bacteria (8.5%). Only subtypes 1, 3 and5 were represented in the 111 archaeal genomes analyzed by the presentinventors. However, the absence of subtypes 2, 4 and 6 from archaealgenomes could be the result of their rarity and the relative paucity ofsequenced archaeal genomes, comprising only 111 out of the 1447 genomesanalyzed.

Taken together, the BREX systems undergo extensive horizontal transfer,with subtype 1 possibly the ancestral form of BREX.

Example 6 Frequent Interruptions in the Adenine-Specific Methylase PglX

One of the type 1 BREX-containing Bacillus subtilis strains obtained wasnot active against any of the tested phages although PCR analysis showedthat it contained the complete BREX system. Upon Illumina whole-genomere-sequencing of the engineered strain, a frameshift mutation in theadenine-specific methylase gene pglX was observed, resulting from asingle nucleotide deletion occurring in a stretch of seven guanine (G)residues at position 2128 (out of 3539 bp) of this gene. These resultsfurther support that the pglX gene is essential for the function of thetype 1 BREX system. Therefore more broadly additional evidence forgenetic variability of pglX in nature was examined.

In 11% (15/135) of the BREX systems that were documented, the pglX genepresented irregularities with respect to the common BREX organization(FIG. 17A). These included seven instances of premature stop codons inthe middle of the gene, two instances of gene duplication and sixoccurrences where a full length pglX gene was adjacent to one or morepartial forms of pglX (with an extreme example in Methacobrevibactersmithii, where five truncated forms of the pglX are found near the fulllength gene (FIG. 17B). The complete and truncated forms of themethylase usually resided on opposite strands and were accompanied by agene annotated as a recombinase, possibly involved in switching betweenthe two versions of pglX. Indeed, when analyzing the genomes of twostrains of Lactobacillus rhamnosus GG that were sequenced independently(NCBI accessions FM179322 (NC_013198 SEQ ID NO: 1) and AP011548(NC_017482 SEQ ID NO: 2), the present inventors found that the pglXsequence was identical between the strains except for a cassette of 313bp that was switched between the full length and truncated pglX genes(FIG. 17C). The interchanged cassette was flanked by two invertedrepeats suggesting a recombination-based cassette switching possiblymediated by the accompanying recombinase.

DNA shuffling via recombination events was previously shown to controlphase variation in bacterial defense-related genes to alter thespecificity or to mitigate toxic effects of specific genes in theabsence of phage pressure⁴⁻²⁶. Taken together, since no other geneexcept for pglX presented such high rates of irregularities, theseresults marked pglX as possibly undergoing frequent phase-variation,suggesting that this gene might confer specificity in the BREX system,or, alternatively, is particularly toxic.

Example 7 Extensive Identification of BREX Systems in ProkaryoticGenomes

Following the initial homology searches in 1447 genomes described indetails hereinabove, the present inventors performed an extensivehomology search on a bigger set of genomes, 2263 complete and 5493 draftgenomes, using the 14 genes associated with BREX systems (Table 1above). Only gene clusters containing the two core genes (pglX andbrxC/pglY) and at least two additional genes were considered (Tables10-16 above).

The homology searches of the BREX genes in the 5493 genomes found 536BREX systems in 9.3% (513/5493) of all genomes analyzed.

BREX type 1, the most common form of BREX, appeared 409 times in 398genomes (Tables 10 and 16 above).

In most cases a single BREX system per organism was found, with only 21(4%) of genomes harboring more than one subtype (Table 16 above).

In addition, in 25% (134/536) of the identified systems, one of thegenes was either missing or has become a pseudogene (Tables 10-15above), possibly representing inactivated systems.

Furthermore, in 11.5% (62/536) of the BREX systems that were documented,the pglX gene presented irregularities with respect to the common BREXorganization.

Taken together, the broader analysis of the 7756 genomes reinforced allfindings obtained with the 1447 set of genomes described hereinabove.

Taken together, the above results described a phage resistance systemwidespread in bacteria and archaea, which the present inventors denotedBREX system. The BREX family of systems can be divided into six coherentsubtypes containing 4-8 genes each, two of which are core genes, pglZand brxC/pglY, present in all systems. The results also suggested pglXmight confer specificity in the BREX system, or, alternatively, isparticularly toxic. Moreover, the BREX systems undergo extensivehorizontal transfer, with subtype 1, the most frequent subtype of thissystem, possibly the ancestral form of BREX.

In addition, the results demonstrated that the BREX type 1 systemconfers complete or partial resistance against phages spanning a widephylogeny of phage types, including lytic and temperate phages, even inthe first cycle of infection. The abundance of this system and theefficiency in which it protects against phages implies that it plays animportant role as a major line of defense encoded by bacteria againstphages.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

TABLE 17 SEQ ID NO. vs. Accession Number Index SEQ ID NO. Accession No.3 643625571 4 640726035 5 640526647 6 643706992 7 649671139 8 3784434549 644886774 10 438000910 11 2506688719 12 2506688721 13 643706994 14326402148 15 344198243 16 169786889 17 407698262 18 86156430 19 5647543220 146351220 21 374998023 22 386866198 23 410470815 24 323524377 25330814956 26 134294128 27 313671969 28 78042616 29 189499000 30374294493 31 374294493 32 300853232 33 302384444 34 302384444 35339441064 36 339324158 37 257057919 38 270307451 39 300087139 4089892746 41 408417460 42 256827818 43 402570638 44 239904639 45 4656212846 387151873 47 297567992 48 401761514 49 385785459 50 387869382 51259906682 52 85372828 53 386632422 54 386627502 55 157159467 56260866153 57 218561636 58 172056045 59 347534971 60 302877245 61302877245 62 239825584 63 400756305 64 332661890 65 386712343 66435852812 67 397655102 68 385816611 69 301065125 70 385812838 71403514032 72 268318562 73 338202359 74 385826720 75 258506995 76296110131 77 148642060 78 397779166 79 409187964 80 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363898451 445 472152144 446 419240736 447 419383938 448424630728 449 374627936 450 374627936 451 418342894 452 420088308 453255743719 454 455929923 455 206975561 456 423164021 457 417522340 458419831962 459 139439021 460 314944229 461 423147095 462 224485442 463422305960 464 225363921 465 417815500 466 425040990 467 343177620 468423878932 469 211593993 470 418398711 471 378759082 472 402296507 473259048327 474 455905617 475 224514842 476 357631901 477 384096966 478425069205 479 265756999 480 427646214 481 403955718 482 373471569 483456039135 484 224993695 485 149109670 486 365096837 487 298527635 488395208219 489 458920171 490 419246266 491 423155496 492 421624890 493427425534 494 458913812 495 417191381 496 424621178 497 262405036 498422890700 499 419229938 500 424015456 501 335043876 502 419894708 503472202862 504 211594035 505 424618529 506 224581212 507 169343945 508405982616 509 469924367 510 417292897 511 418336054 512 423152690 513419841668 514 421338374 515 325917999 516 365822320 517 294645413 518294645413 519 419367985 520 419367985 521 419343475 522 419343475 523365833706 524 365833706 525 448302553 526 448302553 527 448681954 528448681954 529 448413196 530 448413196 531 470888868 532 443475057 533386810750 534 196035064 535 419725778 536 336430981 537 242355593 538397905651 539 390993910 540 323701113 541 288572734 542 325672510 543408373871 544 423394306 545 254434980 546 334130722 547 472439485 548398888999 549 196044104 550 374534854 551 415887008 552 343509531 553381156824 554 374623705 555 326204471 556 326389902 557 256752440 558256752440 559 211606481 560 384563951 561 453074660 562 375098335 563359765453 564 451335404 565 126666487 566 257461537 567 440700728 568163804182 569 458780588 570 383824531 571 224581088 572 383827549 573365878943 574 254173939 575 149176214 576 458859600 577 441515884 578163719735 579 381168746 580 211606481 581 288920043 582 452746574 583319947885 584 2530355709 585 [Vibrio cholerae O1 str. 3582-05] 586650306799 587 373108119 588 373108120 589 2520346679 590 384563951 591453074660 592 375098335 593 359765453 594 451335404 595 126666487 596257461537 597 440700728 598 163804182 599 458780588 600 383824531 601224581088 602 383827549 603 365878943 604 254173939 605 149176214 606458859600 607 441515884 608 163719735 609 381168746 610 211606481 611211606481 612 288920043 613 452746574 614 643625571 615 640726035 616640526647 617 326402593 618 344200467 619 169786942 620 407700180 62186159654 622 56478399 623 403571624 624 374998147 625 386867049 626410471296 627 323527069 628 330816348 629 134296119 630 313673973 63178043274 632 189500610 633 374294726 634 374297213 635 300856435 636302387137 637 302388015 638 310658340 639 339442903 640 339327420 641257060038 642 270307693 643 300088703 644 89893422 645 408420313 646256829113 647 402570961 648 239908251 649 46580432 650 387153155 651297570109 652 401762042 653 385787389 654 387872412 655 259909432 65685375773 657 386637212 658 386632292 659 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296269522 80683646203 807 432329234 808 392407895 809 169830968 810 206896068 811428774400 812 428774405 813 291286510 814 431792782 815 253699434 816327312662 817 148270869 818 396584764 819 458152047 820 472443547 821336440365 822 432379520 823 374625979 824 424979861 825 472222755 826419173682 827 331001731 828 375004439 829 421082306 830 417605263 831332655414 832 373485787 833 417269258 834 363899395 835 419281128 836262371247 837 260887939 838 262374465 839 418024979 840 472199360 841419235503 842 352101112 843 229817501 844 443510724 845 407801756 846424026177 847 443537945 848 329896082 849 433118460 850 307287300 851470894102 852 420093469 853 443475020 854 423730158 855 458790583 856458059910 857 320095081 858 373113625 859 421857673 860 422905804 861421735408 862 293373689 863 424001208 864 410103029 865 301024307 866317501018 867 472217726 868 423288074 869 425019539 870 238756191 871398800471 872 433099194 873 457927366 874 419389186 875 229814945 876422028737 877 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425444980 1021 472183766 1022 424655758 1023 3341256711024 126665995 1025 423709629 1026 437842751 1027 418008631 1028431534713 1029 410637164 1030 363897443 1031 472152152 1032 4192408711033 419383944 1034 424630790 1035 374629429 1036 374630018 1037418342902 1038 420088375 1039 255743727 1040 443518100 1041 2069756171042 423164024 1043 417522346 1044 419832016 1045 139439065 1046314941481 1047 423147141 1048 255014565 1049 422306080 1050 2548509281051 417815511 1052 425040993 1053 340752704 1054 423878986 105590418997 1056 418398870 1057 381199747 1058 402700381 1059 2601015681060 443514286 1061 160944224 1062 358012808 1063 383114787 1064425069876 1065 265757019 1066 427646244 1067 404399332 1068 3734691241069 443534358 1070 225374526 1071 167551250 1072 365096843 1073298528556 1074 395208610 1075 458920176 1076 419246582 1077 4231555041078 421624973 1079 427425586 1080 458913829 1081 417191550 1082424621187 1083 262405137 1084 422890703 1085 419230121 1086 4240155101087 335043947 1088 419894757 1089 472202871 1090 89073847 1091424618575 1092 237744599 1093 169343946 1094 405982586 1095 4317539011096 417292957 1097 418336066 1098 423152698 1099 419841735 1100421338422 1101 325918007 1102 366087502 1103 294645433 1104 2946454341105 419368080 1106 419368151 1107 419343546 1108 419343547 1109365831806 1110 365831811 1111 448302568 1112 448302570 1113 4486820781114 448682080 1115 448413236 1116 448413238 1117 470888966 1118443475072 1119 386811046 1120 196035162 1121 419725844 1122 3364268921123 257885934 1124 397905666 1125 392538885 1126 323701234 1127288575102 1128 325266723 1129 408373945 1130 423398559 1131 2544356281132 334130736 1133 472439490 1134 398889059 1135 196044278 1136374812241 1137 415887047 1138 343509540 1139 381156866 1140 3746237101141 326204498 1142 326389943 1143 256752454 1144 256752455 114588811250 1146 384564491 1147 453074722 1148 375098941 1149 3597655061150 451335435 1151 126666537 1152 291003193 1153 440700790 1154168697899 1155 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What is claimed is:
 1. An expression vector comprising a nucleic acidsequence encoding a functionally active type 1 Bacteriophage Exclusion(BREX) system comprising a pglX polypeptide comprising anadenine-specific methylase domain (pfam13659, COG1002/COG0286), abrxC/pglY polypeptide comprising an ATP binding domain (pfam10923), apglZ polypeptide comprising an alkaline phosphatase domain (pfam08665),a brxL polypeptide comprising a Lon-like protease domain (COG4930), abrxA polypeptide comprising a DUF1819 domain (pfam08849), and a brxBpolypeptide comprising a DUF1788 domain (pfam08747); wherein the aminoacid sequences of each of said type 1 BREX system polypeptides, are setforth as follows: (a) the brxA polypeptide amino acid sequence isselected from the group consisting of SEQ ID NOs: 4953-5064, 5102-5367,5399-5414, and 6225; (b) the brxB polypeptide amino acid sequenceselected from the group consisting of SEQ ID NO: 5570-5686, 5698-5939,5944-5947, 6206, and 6227; (c) the brxC/pglY polypeptide amino acidsequence selected from the group consisting of SEQ ID NO: 615, 617-735,818-1110, 1170-1175, and 6229; (d) a pglX polypeptide amino acidsequence selected from the group consisting of SEQ ID NO: 2766-2916,2954-3251, 3276-3280, 6182, 6190, 6192, 6194, 6196, 6198, and 6231; (e)a pglZ polypeptide amino acid sequence selected from the groupconsisting of SEQ ID NO: 1716-1834, 1905-2192, 2248, 2250, 6204, and6233; and (f) a brxL polypeptide amino acid sequence selected from thegroup consisting of SEQ ID NO: 4028-4298, 4300-4402, 6165, and 6235;said expression vector further comprising a heterologous cis-actingregulatory element for directing expression of said nucleic acidsequence encoding the type 1 BREX system, wherein said type 1 BREXsystem confers phage resistance to a bacterium recombinantly expressingsame, wherein said bacterium does not endogenously express a functionaltype 1 BREX system, and wherein said bacterium is a species selectedfrom the group consisting of a Lactococcus species, a Streptococcusspecies, a Lactobacillus species, a Leuconostoc species, an Oenococcusspecies, a Pediococcus species, a Bifidobacterium species, and aPropionibacterium species.
 2. A phage defense composition, comprising asan active ingredient the expression vector of claim 1, and an acceptablecarrier or diluent.
 3. An isolated, genetically modified bacterium,wherein said bacterium does not express a functional type 1Bacteriophage Exclusion (BREX) system endogenously, modified to expressa functionally active type 1 BREX system that confers phage resistanceto said bacterium, said type 1 BREX system comprising a pglX polypeptidecomprising an adenine-specific methylase domain (pfam13659,COG1002/COG0286), a brxC/pglY polypeptide comprising an ATP bindingdomain (pfam10923), a pglZ polypeptide comprising an alkalinephosphatase domain (pfam08665), a brxL polypeptide comprising a Lon-likeprotease domain (COG4930), a brxA polypeptide comprising a DUF1819domain (pfam08849), and a brxB polypeptide comprising a DUF1788 domain(pfam08747); wherein the amino acid sequences of each of said type 1BREX system polypeptides, are set forth as follows: (a) the brxApolypeptide amino acid sequence is selected from the group consisting ofSEQ ID NOs: 4953-5064, 5102-5367, 5399-5414, and 6225; (b) the brxBpolypeptide amino acid sequence selected from the group consisting ofSEQ ID NO: 5570-5686, 5698-5939, 5944-5947, 6206, and 6227; (c) thebrxC/pglY polypeptide amino acid sequence selected from the groupconsisting of SEQ ID NO: 615, 617-735, 818-1110, 1170-1175, and 6229;(d) a pglX polypeptide amino acid sequence selected from the groupconsisting of SEQ ID NO: 2766-2916, 2954-3251, 3276-3280, 6182, 6190,6192, 6194, 6196, 6198, and 6231; (e) a pglZ polypeptide amino acidsequence selected from the group consisting of SEQ ID NO: 1716-1834,1905-2192, 2248, 2250, 6204, and 6233; and (f) a brxL polypeptide aminoacid sequence selected from the group consisting of SEQ ID NO:4028-4298, 4300-4402, 6165, and 6235; and wherein said bacterium is aspecies selected from the group consisting of a Lactococcus species, aStreptococcus species, a Lactobacillus species, a Leuconostoc species,an Oenococcus species, a Pediococcus species, a Bifidobacterium species,and a Propionibacterium species.
 4. The isolated, genetically modifiedbacterium of claim 3, wherein said bacterium is resistant to: a firstcycle phage infection, phage lysogeny, lytic phage and/or phage DNAreplication.
 5. A method of protecting a bacterium from phage attack,wherein said bacterium does not express a functional type 1Bacteriophage Exclusion (BREX) system endogenously, the methodcomprising: (1) introducing into the bacterium an expression vectorcomprising a nucleic acid sequence encoding a functionally active type 1Bacteriophage Exclusion (BREX) system comprising a pglX polypeptidecomprising an adenine-specific methylase domain (pfam13659,COG1002/COG0286), a brxC/pglY polypeptide comprising an ATP bindingdomain (pfam10923), a pglZ polypeptide comprising an alkalinephosphatase domain (pfam08665), a brxL polypeptide comprising a Lon-likeprotease domain (COG4930), a brxA polypeptide comprising a DUF1819domain (pfam08849), and a brxB polypeptide comprising a DUF1788 domain(pfam08747); wherein the amino acid sequences of each of said type 1BREX system polypeptides, are set forth as follows: (a) the brxApolypeptide amino acid sequence is selected from the group consisting ofSEQ ID NOs: 4953-5064, 5102-5367, 5399-5414, and 6225; (b) the brxBpolypeptide amino acid sequence selected from the group consisting ofSEQ ID NO: 5570-5686, 5698-5939, 5944-5947, 6206, and 6227; (c) thebrxC/pglY polypeptide amino acid sequence selected from the groupconsisting of SEQ ID NO: 615, 617-735, 818-1110, 1170-1175, and 6229;(d) a pglX polypeptide amino acid sequence selected from the groupconsisting of SEQ ID NO: 2766-2916, 2954-3251, 3276-3280, 6182, 6190,6192, 6194, 6196, 6198, and 6231; (e) a pglZ polypeptide amino acidsequence selected from the group consisting of SEQ ID NO: 1716-1834,1905-2192, 2248 2250, and 6204, and 6233; and (f) a brxL polypeptideamino acid sequence selected from the group consisting of SEQ ID NO:4028-4298, 4300-4402, 6165, and 6235; and (2) expressing in thebacterium said functional type 1 BREX system from said expressionvector; wherein upon phage attack, said functional type 1 BREX systemconfers phage resistance to the bacterium and thereby protects thebacterium from phage attack; wherein said expression vector comprises aheterologous cis-acting regulatory element for directing expression ofsaid nucleic acid sequence; and wherein said bacterium is a speciesselected from the group consisting of a Lactococcus species, aStreptococcus species, a Lactobacillus species, a Leuconostoc species,an Oenococcus species, a Pediococcus species, a Bifidobacterium species,and a Propionibacterium species.
 6. The method of claim 5, wherein saidphage is selected from the group consisting of SPβ, SP16, Zeta, Φ3T andSPO2.
 7. The method of claim 5, wherein said type 1 BREX system does notconfer resistance to phages Φ105, rho10 and rho14.
 8. The method ofclaim 5, wherein said phage is a lytic phage.
 9. An isolated bacteriumcomprising a nucleic acid sequence encoding a functionally active type 1Bacteriophage Exclusion (BREX) system, said type 1 BREX system expressedfrom a plasmid or a transposon, wherein said isolated bacterium does notendogenously express said type 1 BREX system, wherein said functionallyactive type 1 BREX system confers phage resistance to the bacteriumrecombinantly expressing same, and wherein said type 1 BREX systemcomprises a pglX polypeptide comprising an adenine-specific methylasedomain (pfam13659, COG1002/COG0286), a brxC/pglY polypeptide comprisingan ATP binding domain (pfam10923), a pglZ polypeptide comprising analkaline phosphatase domain (pfam08665), a brxL polypeptide comprising aLon-like protease domain (COG4930), a brxA polypeptide comprising aDUF1819 domain (pfam08849), and a brxB polypeptide comprising a DUF1788domain (pfam08747); wherein the amino acid sequences of each of saidtype 1 BREX system polypeptides, are set forth as follows: (a) the brxApolypeptide amino acid sequence is selected from the group consisting ofSEQ ID NOs: 4953-5064, 5102-5367, 5399-5414, and 6225; (b) the brxBpolypeptide amino acid sequence selected from the group consisting ofSEQ ID NO: 5570-5686, 5698-5939, 5944-5947, 6206, and 6227; (c) thebrxC/pglY polypeptide amino acid sequence selected from the groupconsisting of SEQ ID NO: 615, 617-735, 818-1110, 1170-1175, and 6229;(d) a pglX polypeptide amino acid sequence selected from the groupconsisting of SEQ ID NO: 2766-2916, 2954-3251, 3276-3280, 6182, 6190,6192, 6194, 6196, 6198, and 6231; (e) a pglZ polypeptide amino acidsequence selected from the group consisting of SEQ ID NO: 1716-1834,1905-2192, 2248 2250, and 6204, and 6233; and (f) a brxL polypeptideamino acid sequence selected from the group consisting of SEQ ID NO:4028-4298, 4300-4402, 6165, and 6235; wherein said bacterium is aspecies selected from the group consisting of a Lactococcus species, aStreptococcus species, a Lactobacillus species, a Leuconostoc species,an Oenococcus species, a Pediococcus species, a Bifidobacterium species,and a Propionibacterium species.